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Structure-Guided Functional Characterization of Enediyne Self-Sacrifice Resistance Proteins, CalU16 and CalU19

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† Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States
‡ Center for Pharmaceutical Research and Innovation (CPRI), College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States
§ Department of Chemistry and Biochemistry, Northeast Structural Genomics Consortium, Miami University, Oxford, Ohio 45056, United States
∥ Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, New York 10027, United States
⊥ Department of Molecular and Cellular Biochemistry & Center for Structural Biology, College of Medicine, University of Kentucky, Lexington, Kentucky 40536, United States
# Complex Carbohydrate Research Center, Northeast Structural Genomics Consortium, University of Georgia, Athens, Georgia 30602, United States
∇ Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, United States
○ Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, United States
◆ Department of Biochemistry, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, United States
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
*Email: [email protected]
*Email: [email protected]
Cite this: ACS Chem. Biol. 2014, 9, 10, 2347–2358
Publication Date (Web):July 31, 2014
https://doi.org/10.1021/cb500327m
Copyright © 2014 American Chemical Society
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Abstract

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Calicheamicin γ1I (1) is an enediyne antitumor compound produced by Micromonospora echinospora spp. calichensis, and its biosynthetic gene cluster has been previously reported. Despite extensive analysis and biochemical study, several genes in the biosynthetic gene cluster of 1 remain functionally unassigned. Using a structural genomics approach and biochemical characterization, two proteins encoded by genes from the 1 biosynthetic gene cluster assigned as “unknowns”, CalU16 and CalU19, were characterized. Structure analysis revealed that they possess the STeroidogenic Acute Regulatory protein related lipid Transfer (START) domain known mainly to bind and transport lipids and previously identified as the structural signature of the enediyne self-resistance protein CalC. Subsequent study revealed calU16 and calU19 to confer resistance to 1, and reminiscent of the prototype CalC, both CalU16 and CalU19 were cleaved by 1in vitro. Through site-directed mutagenesis and mass spectrometry, we identified the site of cleavage in each protein and characterized their function in conferring resistance against 1. This report emphasizes the importance of structural genomics as a powerful tool for the functional annotation of unknown proteins.

The calicheamicins are a prototype of the naturally occurring 10-membered enediyne antitumor antibiotic family and were first reported in 1989 as metabolites of Micromonospora echinospora spp. calichensis. (1-3) Members of this family share a structurally conserved bicyclo[7.3.1]enediyne core, also often referred to as the “warhead” as this structural unit is central to the fundamental enediyne mechanism of action (Figure 1A). (4-6) In all family members, the enediyne core is strategically decorated with a bioorthogonal “triggering” system and specific appendages that enhance affinity to the metabolite’s target (DNA/RNA). The calicheamicin trigger system (and that of esperamicins, shishijimicins, and namenamicins) is comprised of a unique trisulfide that, upon reduction by bioreductants such as glutathione, induces an intramolecular hetero-Michael addition at C-9 (Figure 1B). (7-9) The resulting fused enediyne core ring strain invokes a spontaneous cycloaromatization reaction which proceeds via a highly reactive diradical intermediate that is rapidly quenched by any suitable hydrogen source. (7, 10) Calicheamicin’s high affinity for the minor groove of DNA, by virtue of its aryltetrasaccharide, insures that the diradical species is quenched via hydrogen abstraction from the backbone of opposing strands of dsDNA to form DNA radicals that, in the presence of oxygen, result in facile double-strand scission. (11-13) The study of calicheamicin’s many fascinating architectural and functional facets have led to numerous discoveries/advances in chemistry, (14, 15) enzymology, (16-26) and anticancer drug development (27, 28) over the past three decades. Yet, while the gene cluster encoding for calicheamicin biosynthesis was cloned from M. echinospora and sequenced nearly a decade ago, (16) there remain a number of genes (∼30%) within this locus annotated as “unknowns” due to a lack of homologues and/or biochemical characterization of corresponding gene products.

Figure 1

Figure 1. (A) Selected structures of naturally occurring 10-membered enediynes. The “warhead” is highlighted in red. (B) Proposed mechanism of cycloaromatization of 1 and its effect on DNA scission and CalC self-sacrifice mechanism.

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Herein, we describe the application of structural genomics as a basis for the functional characterization of two proteins encoded by such “unknowns”—CalU16 and CalU19. Specifically, structure elucidation (via both NMR and X-ray crystallography) revealed CalU16 to be a structural homologue of CalC, a protein previously characterized as among the first reported enediyne “self-sacrifice” resistance proteins. (18, 19) Prompted by this structure-based revelation, subsequent biochemical characterization of CalU16 and its homologue CalU19 revealed both to serve in a similar capacity wherein CalU19 also displayed the unprecedented ability to trigger enediyne cycloaromatization in the absence of endogenous reducing agents. Cumulatively, this work extends the body of work focused upon understanding how bacteria construct and control highly reactive and lethal metabolites and expands the number of known “self-sacrifice” enediyne resistance proteins. This work also serves to illustrate the impact of structure determination as a critical tool for the functional annotation of unassigned genes.

Results and Discussion

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CalU16 Structure and CalU19 Homology Model

Protein Structure Initiative (PSI- Biology) employs a complementary combination of NMR spectroscopy and/or X-ray crystallography to determine 3D-structures of biologically relevant targets. The structure of CalU16 was solved both by X-ray crystallography (Protein Database Bank (PDB: 4FPW)) and NMR (PDB: 2LUZ). The crystal structure of CalU16 was determined at 2.50 Å resolution, refined to an Rcryst and Rfree of 24.1% and 28.2% (Table 1), respectively, and is comprised of two subunits in an asymmetric unit belonging to space group P61 (Figure 2). Of the 182 residues of CalU16, the first 4 residues, the last 2 residues, and a dynamic loop containing 19 residues (a.a. 141–159) from subunit A and 15 residues (a.a. 144–158) from subunit B were not modeled due to insufficient electron density. Structure and quality statistics are given in Table 1. The corresponding solution structure of CalU16 was determined by multidimensional heteronuclear NMR spectroscopy (Supporting Information Figures S1–S3). Nearly complete 1H, 15N, and 13C assignments were obtained and structures were calculated from residual dipolar couplings (RDCs), chemical shift-derived dihedral angle constraints, and Nuclear Overhauser Effect (NOE)-derived distance constraints via standard protocols. Structure and quality statistics are given in Table 2 for the ensemble of 20 lowest energy structures, indicating the high quality of the structure. Intrinsic disorder was observed for the first 6 residues, the last 3 residues, and 21 residues that includes a loop and part of the following helix (a.a. 143–163) and is corroborated by a lack of long-range NOEs and low (<0.6) 15N{1H} heteronuclear values (Supporting Information Figure S3).

Figure 2

Figure 2. Structure of CalU16. (A) Overlay of NMR (green) and monomers of the crystal (brick- red) structures of CalU16. The N- and C-termini of the protein are labeled, while the dynamic loop is colored yellow. (B) Monomer with secondary structural elements labeled. The residues in the hydrophobic cavity are represented as stick models. (C) B-factors for the Cα atoms in the crystal structure (left) and Cα RMSD values from the NMR ensemble (right) are mapped to the color and tube diameter of “putty” traces showing the general agreement (correlation coefficient 0.559) between the structures.

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Table 1. Summary of Crystal Parameters, Data Collection, and Refinement Statisticsa
propertyvalue
Crystal Param.
space groupP61
molecules per asymmetric unit2
VM (Å3 Da–1)2.95
unit-cell param. (Å)a = b = 51.85, c = 305.70
Data Collection Statistics
wavelength (Å)0.9790
resolution range (Å)50.00–2.50 (2.59–2.50)
no. of reflections (measured/unique)236417/16051
completeness (%)99.9 (99.5)
Rmergeb0.056 (0.169)
redundancy14.8 (12.3)
mean I/σ (I)27.4 (10.4)
Refinement and Model Statistics
resolution range (Å)44.91–2.50
no. of reflections (work/test)15160/794
Rcrystc0.241 (0.325)
Rfreed0.282 (0.371)
twin operatorK, H, -L
twin fraction0.50
RMSD bonds (Å)0.009
RMSD angles (deg)1.453
B factor (protein/solvent) (Å2)53.8/50.9
no. of protein atoms2497
no. of waters128
Ramachandran Plot (%)e
most favorable regions91.3
allowed regions5.8
disallowed regions2.9
Global Quality Scores (Raw/Z-Score)
Verify3D0.4/–1.0
ProsaII0.5/–0.6
Procheck G factor (phi-psi)–0.6/–2.0
Procheck G factor (all)–0.5/–2.7
Molprobity clash score9.6/–1.8
PDB ID4FPW
a

Values in parentheses are for the highest resolution shell.

b

Rmerge = ∑h ∑i | Ii(h) – ⟨I(h)⟩|/∑h∑iIi(h), where Ii(h) is the intensity of an individual measurement of the reflection and ⟨I(h)⟩ is the mean intensity of the reflection.

c

Rcryst = ∑h ||Fobs| – |Fcalc||/∑h |Fobs|, where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively.

d

Rfree was calculated as Rcryst except that it uses 10% of the reflection data omitted from refinement.

e

Calculated using Molprobity. (71)

Table 2. Summary of CalU16 Solution Structural Statisticsa
propertyvalue
Completeness of Resonance Assignmentsb
backbone (%)98.3
side chain (%)97.9
aromatic (%)96.2
stereospecific methyl (%)85.8
Conformationally Restricting Constraintsc
distance constraints 
total2522
intraresidue (i = j)471
  sequential (|I – j| = 1)614
medium range (1 < |i – j| < 5)458
long-range (|i – j| ≥ 5)979
dihedral angle constraints222
hydrogen bond constraints192
NH RDC constraints (gel NH/NC′)75/59
no. of constraints per residue16.5
no. of long-range constraints per residue6.0
Residual Constraint Violationsc
avg. no. of distance violations per structure
0.1–0.2 Å24.8
0.2–0.5 Å6.2
> 0.5 Å0
avg no. of dihedral angle violations per structure
1–10°17.8
> 10°0.2
RDC Qrmsd (gel NH/NC′)d0.4/0.3
Model Qualityc
RMSD backbone atoms (Å)e0.7
RMSD heavy atoms (Å)e1.2
RMSD bond lengths (Å)0.018
RMSD bond angles (deg)1.3
MolProbity Ramachandran statisticsc,e 
most favored regions (%)94.6
allowed regions (%)5.4
disallowed regions (%)0
global quality scores (Raw/Z-score)c
Verify3D0.4/–0.3
ProsaII0.5/–0.6
Procheck G factor (φ–ψ)e–0.4/ −1.3
Procheck G factor (all)e–0.3/–2.0
MolProbity clash score16.0/–1.2
RPF scoresf 
recall/precision0.98
F-measure/DP-score0.94
Model Contents
ordered residue rangee7–65, 71–89, 95–140, 161–179
BMRB accession no.18547
PDB ID2LUZ
a

Structural statistics computed for the ensemble of 20 deposited structures.

b

Computed using AVS software from the expected number of resonances, excluding: highly exchangeable protons (N-terminal, Lys, and Arg amino groups, hydroxyls of Ser, Thr, Tyr), carboxyls of Asp and Glu, nonprotonated aromatic carbons, for residues 1–182.

c

Calculated using PSVS 1.4. Average distance violations were calculated using the sum over r–6.

d

RDC goodness-of-fit quality factor Qrmsd determined using PALES.

e

Based on ordered residue ranges [S(φ) + S(ψ) > 1.8].

f

RPF scores reflecting the goodness-of-fit of the final ensemble of structures (including disordered residues) to the NOESY data and resonance assignments.

Superposition of the CalU16 solution structure with individual subunits of the crystal structure reveals a rmsd of 0.88 Å, indicating that the structures are quite similar. The major difference between the two structures is the absence of the previously mentioned dynamic loop region (residues 141–159) in the crystal structure (Figure 2). Interestingly, the crystal structure of CalU16 contains two subunits in the asymmetric unit related by 2-fold noncrystallographic symmetry, with a small contact interface (calculated to be ∼530 Å2 by PISA). (29) Based on analytical static light scattering in-line (Supporting Information Figure S4) with gel filtration chromatography and NMR correlation time estimates, CalU16 exists as a monomer in solution. The final difference map also reveals an additional long tubular feature in hydrophobic pocket, perhaps representing a bound unknown E. coli metabolite carried through the purification procedure (Supporting Information Figure S5).
The CalU16 monomer folds into a globular domain formed by four α-helices and seven antiparallel β-strands in the order: β1-β2-α1-α2-β3-β4-β5-β6-β7-α3-loop-α4 (Figure 2). The domain exhibits a common tertiary structure consisting of a helix-grip-fold, characteristic of STeroidogenic Acute Regulatory protein related lipid Transfer (START) domains implicated in possessing a hydrophobic cavity for ligand binding. (30, 31) The hydrophobic cavity in CalU16 spans the entire length of CalU16, formed by seven β-strands and three helices, α1, α2, and α3, encompassing residues Ile24, Ile26, Val37, Trp38, Ile47, Trp50, Phe51, Ile52, Phe64, Leu66, Leu83, Ile74, Ile85, Trp87, Val97, Leu99, Leu101, Leu110, Leu112, Val125, Trp129, Leu133, Leu136, and Ile140 (Figure 2). In addition to the START domain core, the fourth helix (α4) of CalU16 contributes an additional structural unit. Such extra secondary structural elements are common upon START domain members where they contribute to a range of structural/functional roles. (19, 32)
CalU19 is another protein of unknown function encoded by a gene in the calicheamicin gene cluster, which displays 42% sequence identity to CalU16 (Supporting Information Figure S6). A CalU16-based homology model of CalU19, generated using SWISS-MODEL also highlights the presence of a signature hydrophobic core consistent with START domain family members, implicating a similar function (Supporting Information Figure S7).

Structurally-Related Proteins

A structure-based similarity search of CalU16 using DaliLite server (33) and PDBeFold (34) returned >100 hits that share close structural similarity. Figure 3 and Supporting Information Table S1 summarize a small selection of functionally characterized structural homologues of CalU16 (Figure 3A). Among these is CalC (PDB: 2L65) (Figure 3B), the prototype calicheamicin self-sacrifice resistance protein encoded by the gene calC from the same biosynthetic gene cluster as CalU16. (19) In addition, three structures (Figure 3C–E) associated with other natural product biosynthetic pathways were identified. Specifically, TcmN Aro/Cyc (PDB: 3TVQ) is a polyketide aromatase/cyclase (Figure 3C) involved in the regiospecific cyclization of tetracenomycin, (35) Hyp-1 (PDB: 3IE5) is involved in the formation of hypericin (Figure 3D), (36) while NCS (PDB: 2VQ5) is involved in the biosynthesis of (S)-norcoclaurine (Figure 3E). (37) Other representative structural homologues identified include plant allergen Bet v1-J (PDB: 4A8U), (38) abscisic acid receptor (PDB: 4N0G), (39) cytokinin-specific binding protein (PDB: 2FLH), (40) and major celery allergen protein (PDB: 2BK0) (Supporting Information Table S1). (41) A structural alignment of CalU16 with these homologues highlights the striking conservation of the core structural fold, which contributes to the binding site for a structurally diverse array of ligands including steroids, lipids, hormones, and polyketides. However, a clear structural difference in CalU16 is the presence of an additional helical region near the residues 160–180 (α4 helix as shown in Figure 2B). While this extra helix is not observed in the other homologues of known function, two functionally uncharacterized CalU16 structural homologues also have this helix (PDB: 2NN5 and 2K5G). (42, 43) Despite the plethora of available sequences and structures, (31, 44) START domain structure/sequence alone remains insufficient to assign function for newly discovered family members. Thus, additional structures and parallel biochemical characterization are needed to establish broader structure–activity relationships of START domain proteins.

Figure 3

Figure 3. CalU16 structural homologues. (A) CalU16 (PDB: 4FPW); (B) CalC (PDB: 2L65), calicheamicin resistance protein; (C) TcmN Aro/Cyc (in complex with trans-dihydroquercetin; PDB: 3TVQ) involved in the biosynthesis of tetracenomycin; (D) Hyp-1 (in complex with ethylene glycol; PDB: 3IE5) involved in the biosynthesis of hypericin; (E) NCS (in complex with hydroxybenzaldehyde; PDB: 2VQ5) involved in the biosynthesis of norcoclaurine.

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Heterologous Expression of calU16 or calU19 in E. coli Confers Resistance to Calicheamicin

Our initial cloning of the 1 biosynthetic gene cluster was facilitated by employing a selection for 1 resistance using a M. echinospora cosmid library in E. coli. (16) From positive cosmids identified, an iterative subcloning and selection process ultimately led to the discovery of calC gene and subsequent elucidation of the CalC mechanism. (18) It is important to note that the initial screen for resistance genes relied upon the native Micromonospora promoters for heterologous expression in E. coli, and thus, calU16 and calU19 could have been missed simply due to poor heterologous expression in E. coli. However, the ability of calC (and presumably other self-sacrifice resistance protein encoding genes) to confer resistance upon E. coli provides a convenient indicator for putative function. As a preliminary assessment of potential CalU16/19 function, the genes encoding each protein were expressed in E. coli and the corresponding recombinant strains tested for 1 resistance in a manner reminiscent to that previously reported for CalC. (18) To do so, calU16 and calU19 were cloned into pET28a to provide pSECalU16-E. coli and pSECalU19-E. coli, respectively, and the corresponding heterologous production levels of both CalU16 and CalU19 in E. coli were confirmed via SDS-PAGE to be comparable (Supporting Information Figure S8). A subsequent disc diffusion assay was used to test for 1 resistance of pSECalU16-E. coli, pSECalU19-E. coli, pET28a-E. coli (an empty vector negative control), and pJB2011-E. coli (a calC-expressing positive control). (18) Figure 4 illustrates the rank order of in vivo resistance to be CalC ≈ CalU19 > CalU16 with no resistance in the empty vector-containing negative control. Consistent with this qualitative assessment, the determination of minimum inhibitory concentration (MIC) (Table 3, Supporting Information Figure S9), revealed both pJB2011-E. coli and pSECalU19-E. coli to be 330-fold more tolerant to 1 than the pET28a-E. coli empty vector control while pSECalU16-E. coli was 80-fold more resistant than the control strain. Importantly, this supports the contention that CalU16/19 function in a manner similar to CalC. To further confirm that the 1-resistance pattern of CalC, CalU16, and CalU19 is specific and not an artifact related to general proteins encoded by genes in the calicheamicin gene cluster, an identical study using the unknown-encoding gene calU17 revealed no resistance toward 1 (Supporting Information Figure S10). It is also important to note that 1 does not indiscriminately cleave other proteins such as bovine serum albumin, illustrating the importance of the START domain hydrophobic core for enediyne binding and functional enediyne resistance. (18)

Figure 4

Figure 4. CalU16 and CalU19 assays. Serial disc dilutions of 1 against (A) pSE28a-E. coli (control), (B) pSECalU16-E. coli (CalU16), (C) pSECalU19-E. coli (CalU19), (D) pJB2011-E. coli (CalC). Amount of 1 on discs 1–6 are 10 μg, 1 μg, 100 ng, 50 ng, 10 ng, and 1 ng, respectively. Coomassie-stained 8–12% SDS-PAGE gradient gel of (E) CalU16 and (F) CalU19 in the presence of DTT (lane 1), 1 (lane 2), and DTT and 1 (lane 3).

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Table 3. Minimum Inhibitory Concentration of Strains Used in This Study Against 1
strainMIC (μM)
pSE28a-E. coli0.036
pSECalU16-E. coli3
pSECalU19-E. coli12
pJB2011-E. coli12
pSEU16G128V-E. coli0.36
pSEU16G128R-E. coli0.18
pSEU16G142V-E. coli3
pSEU16G142R-E. coli3
pSEU19G177V-E. coli6
pSEU19G177R-E. coli0.36
pSEU19G181V-E. coli12
pSEU19G191V-E. coli12
pSEU19G196V-E. coli12
pSEU19G206V-E. coli12

CalU16 Biochemical Characterization

The prototype self-sacrifice protein CalC functions via binding calicheamicin and providing an alternative hydrogen source for quenching the highly reactive diradical species formed upon calicheamicin cycloaromatization. In competition assays using the real-time fluorescence-based molecular break light assay, (13) CalC was found to out-compete DNA for calicheamicin and thereby prevent calicheamicin-induced strand scission. In the presence of CalC, the calicheamicin diradical species abstracts the Cα hydrogen of G113 to generate a protein radical that, in the presence of oxygen, leads to proteolysis into two distinct CalC fragments (Figure 1B). (18) Thus, a similar series of proteolysis experiments were conducted to determine whether CalU16 utilizes an analogous mechanism. Figure 4 highlights the outcome of this analysis using N-His6-CalU16. Importantly, incubation of CalU16 with 1 alone led to no reaction, incubation of CalU16 and 1 in the presence of reducing agent (dithiothreitol, DTT) led to the specific cleavage of CalU16 into two fragments of estimated size 16 kDa and 6 kDa based upon SDS-PAGE (Figure 4E). LC-MS/MS analysis of the tryptic digestion of the isolated fragments identified two nontryptic peptides flanking Gly128: 101LSEEGDGTLLELEHATTSEQMLVEVG127V(−NH2) and 129WEMALDFLGMFI141R (see MS/MS spectra of the two peptides in Supporting Information Figure S11). The first peptide ends with a nontryptic residue 127V with an amine group (−NH2) from the 128Gly residue attached. The second peptide starts with 129W further supports that Gly128 is likely the cleavage site due to hydrogen abstraction. In addition, the LC-MS/MS data suggested 142G as another potential cleavage site, based upon a weak peptide signal [129WEMALDFLGMFI141R(−NH2), ion score =24]. However, the corresponding peptide starting with 143D was not identified. Nevertheless, we included both Gly128 and Gly142 in CalU16 as putative sites in the subsequent mutational study to further determine which glycine is the critical point of hydrogen abstraction.
To elucidate the key CalU16 glycine residue that serves as the putative hydrogen donor, four targeted CalU16 glycine mutants (G128V, G128R, G142V, and G142R) were created and tested both in vivo and in vitro. Strains expressing CalU16ΔG142 mutations (pSEU16G142V-E. coli and pSEU16G142R-E. coli) retained wtCalU16 (pSECalU16-E. coli) resistance levels to 1 while mutation of Gly128 (pSEU16G128V-E. coli and pSEU16G128R-E. coli) reduced or abolished tolerance to 1 (Figure 5, Supporting Information Figure S12, Table 3). Consistent with this, 1-based proteolysis of N-His6-CalU16 mutant proteins (Supporting Information Figure S13) revealed CalU16G142V and CalU16G142R to cleave in an identical manner to wtCalU16 in the presence of 1 and DTT while CalU16G128V and CalU16G128R were resistant to cleavage under identical conditions. Cumulatively, this data is consistent with CalU16 Gly128 as the key hydrogen donor in self-sacrifice mechanism, reminiscent of CalC Gly113, wherein cleavage of CalU16 (or CalC) inactivates 1 in a stoichiometric manner.

Figure 5

Figure 5. Site directed mutagenesis of CalU16 and CalU19. Docking models of (A) CalC (B) CalU16, and (C) CalU19 with mutated glycine residues represented as spheres where colored Gly residues indicate cleavage sites, wheat Gly residues indicate mutations that did not affect activity and calicheamicin (1) is represented as a stick model. Results of disc diffusion assay in CalU16 mutants (D) pSE28a-E. coli (control); (E) pSEU16G128V-E. coli; (F) pSEU16G128R-E. coli; (G) pSECalU16-E. coli; and CalU19 mutants (H) pSE28a-E. coli (control); (I) pSEU19G177V-E. coli; (J) pSEU19G177R-E. coli; (K) pSECalU19-E. coli. Amount of 1 on discs 1–6 are 10 μg, 1 μg, 100 ng, 50 ng, 10 ng, and 1 ng, respectively.

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CalU19 Biochemical Characterization

To determine whether the CalU16 homologue CalU19 presents a 1 self-sacrifice resistance function analogous to CalC and CalU16, the expressed N-His6-CalU19 was purified and incubated with 1 alone or in the presence of a reducing agent (DTT). In this study, CalU19 was also cleaved into two fragments by 1 but, in stark contrast to CalC and CalU16, 1-induced CalU19 cleavage occurred even in the absence of the reducing agent DTT (Figure 4F). The estimated sizes of the corresponding CalU19 fragments were ∼21 and 6 kDa based upon SDS-PAGE (Supporting Information Table S2). LC-MS/MS analysis of the chymotrypsin digestion of the isolated fragments identified two nonchymotryptic peptides flanking Gly177: 142RLTPSGDATVLELEHAPVPAEIIPNAAPGAWGIG176A(−NH2) and 178WEMGLVALDDYLAGTLPEGRAVD201W (see MS/MS spectra of the two peptides in Supporting Information Figure S14). The first peptide ends with a nonchymotryptic residue 176A with an amine group (−NH2) from the 177Gly residue attached. The second peptide starts with 178W further supports that Gly177 is likely the cleavage site due to hydrogen abstraction.
To confirm the key CalU19 glycine residue, two targeted CalU19ΔGly177 mutants (G177V and G177R) were created and tested both in vivo and in vitro. To rule out any other possible glycine residues that might be involved in the protein cleavage, four additional glycine residues (Gly181, Gly191, Gly196 and Gly206) were mutated and the corresponding mutants tested in parallel. The E. coli strains expressing CalU19G181V, CalU19G191V, CalU19G196V, CalU19G206V (pSEU19G181V-E. coli, pSEU19G191V-E. coli, pSEU19G196V-E. coli, and pSEU19G206V-E. coli, respectively) retained wtCalU19 (pSECalU19-E. coli) resistance levels to 1 while mutation of Gly177 (pSEU19G177V-E. coli and pSEU19G177R-E. coli) reduced or abolished tolerance to 1 (Figure 5, Supporting Information Figure S15, and Table 3) as depicted by disc diffusion and MIC assays. Consistent with this, 1-based proteolysis of N-His6-CalU19 mutant proteins revealed CalU19G177V and CalU19G177R to be resistant to cleavage when compared with wtCalU19 under identical conditions (Supporting Information Figure S16). In aggregate, this data is consistent with CalU19 Gly177 as the key hydrogen donor in the context of 1 inactivation and served as a basis for modeling the interactions of 1 with CalU16 and CalU19. Notably, the modeled orientation of 1 bound to CalU16 or CalU19 appears to be different from 1 with CalC (Figure 5A–C) consistent with prior observations that the late stage biosynthetic intermediates en route to 1 bind the glycosyltransferases involved in 1 maturation via distinct orientations. (45)
Distinct from CalC and CalU16, CalU19 functioned in the absence of a reducing agent–implicating a residue within CalU19 as a putative reductive activator of the process. CalU19 contains two cysteines (Cys25 and Cys120) where the CalU19 homology model suggests Cys25 to potentially be closer to the trisulfide of 1 in a ligand-bound model than Cys120 (estimated distance 14.80 Å, Supporting Information Figure S17). To test the putative role of Cys25 and Cys120, the corresponding alanine mutants were generated and tested in vivo and in vitro. Surprisingly, no differences in DTT-dependence were observed between wtCalU19, CalU19Cys25A, CalU19Cys120A and the double mutant CalU19Cys25A/Cys120A (Supporting Information Figure S18), suggesting 1 activation via CalU19 occurs via a nonredox mechanism. Notably, cycloaromatized calicheamicin ε (Figure 1B) was detected in all CalU19 proteolysis experiments (including those with the double mutant CalU19Cys25A/Cys120A), indicating there is an alternative CalU19 contributor to trisulfide reductive initiation event.

Enediyne Specificity of CalU16 and CalU19

To test whether CalU16 and CalU19 confer resistance against other 10-membered enediynes, in vivo and in vitro studies similar to those described in the previous sections were performed where 1 was substituted with dynemicin (2) or esperamicin A1 (3). CalU16 and CalU19 each failed to cause any cross resistance against 2 or 3in vivo (Supporting Information Figure S19), and consistent with these observations, neither protein could be cleaved via 2 or 3 in the absence or presence of reducing agent (Supporting Information Figure S20). Thus, CalU16 and CalU19 appear to display enediyne specificity similar to CalC. (18)

Conclusions

High throughput sequencing and genomics are powerful tools to implicate putative function but are still limited by three primary liabilities: (i) functional misannotation due to mistakes propagated throughout large sequence databases; (ii) functional misannotation due to the fact that even highly homologous proteins can present dramatically distinct mechanisms/functions; and (iii) a lack of suitably characterized homologues within existing large sequence databases. (46-48) Structural genomics can augment the use of sequence homology for functional annotation by presenting opportunities to identify structural homologues even where sequence homology may be too low to identify suitable homologues. (49-51) The current study highlights the power of structural genomics to inform putative function as the basis for subsequent biochemical characterization/confirmation and, in doing so, reveals two new enediyne self-sacrifice proteins (CalU16 and CalU19) encoded by genes in the calicheamicin biosynthetic gene cluster. The elucidation of these genes serve as potential new genetic markers which, in conjunction with the signature minimal enediyne PKS cassette, may facilitate future enediyne discovery via genome mining. (17)
While there exist many natural product biosynthetic loci that also encode for more than one resistance mechanism for the encoded natural product, (52, 53) the encoded redundancy highlighted by the current study is uncommon and may suggest discrete self-sacrifice proteins to contribute to subtle distinctions in their localization and/or function. For example, the predicted isoelectric point of CalC (10.16) is dramatically different from CalU16 (4.24) or CalU19 (4.69) (Supporting Information Table S2) and is consistent with CalC’s demonstrated ability to bind DNA (the target of enediynes) under physiological pH. (19) Under identical conditions, CalU16/U19 are predicted to possess an overall negative charge, which may contribute to distinct intracellular localization and/or unique protein–protein interactions possibly including those proteins/enzymes involved in 1 biosynthesis.
The enediyne self-sacrifice genes and corresponding encoded proteins have been validated in the context of conferring enediyne resistance, but it remains possible that such proteins or their proteolytic fragments could also serve alternative roles in Micromonospora. For example, many members of the START domain family function as molecular chaperones as exemplified by the Coq10 homologue CC1736, responsible for transporting ubiquinone to or within the respiratory chain complexes and contributes to dramatically higher biosynthetic efficiency. (54) Although the precedent for molecular chaperones in the context of the structurally related 9-membered enediynes is well established, (5, 55) it remains to be determined whether such proteins play additional roles beyond stabilizing the highly reactive 9-membered enediyne chromophore. Preliminary evidence, based upon the location of the key glycines in CalC, CalU16, and CalU19, suggest that CalC binds 1 in a manner distinct from CalU16/CalU19 which, in the context of a molecular chaperone model, suggests different regions of the cargo would be accessible to putative associated partner proteins (Figure 5A–C). While CalC homologues lack apparent polyketide aromatase/cyclase TcmN Aro/Cyc catalytic residues based upon sequence (Supporting Information Figure S21) and structural (Supporting Information Figure S22) alignments, this relationship raises the intriguing possibility of CalC homologues as the long sought after contributors to enediyne polyketide core folding/cyclization. Indeed, the surface area of the hydrophobic cavities of CalU16 (3986 Å2), CalC (3931 Å2), and TcmN ARO/CYC (3655 Å2) are similar (Supporting Information Figure S23), the latter of which accommodates large (≤C20) linear polyketides. (35) Alternatively, certain proteolytic fragments from CalC, CalU16, and/or CalU19 could also potentially serve as intracellular signals of enediyne concentrations, in a manner conceptually similar to the vancomycin- or β-lactam-inducible three-component regulatory systems, where hydrolytic fragments of the bacterial cell wall serve as key inducers. (56, 57) Studies are underway to further probe whether the functions of START domain congeners encoded by the calicheamicin gene cluster extend beyond simple protection of host against these highly reactive and toxic metabolites.

Methods

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Strains, Materials, and General Methods

All primers (Integrated DNA Technology) and strains used in this study are summarized in Supporting Information Tables S3 and S4, respectively. M. echinospora strain LL6000 and calicheamicin γ1I were graciously provided by Pfizer. Dynemicin and esperamicin A1 were generously provided by Bristol-Myers Squibb. All other reagents and chemicals were purchased from Sigma-Aldrich, unless otherwise stated. All protein structures were illustrated using PyMOL. (58) Gene alignments and analyses were performed using Geneious Pro 5.0.3. (59)

Parental Plasmids for Protein Production

For biochemical characterization, genomic DNA was extracted from M. echinospora LL6000 using the InstaGene Matrix Kit (BioRad) following manufacturer’s protocol. Primers CalU16-NdeI-F/CalU16-HindIII-R, CalU17-NdeI-F/CalU17-HindIII-R, and CalU19-NdeI-F/CalU19-HindIII-R (Supporting Information Table S3) were used to amplify each of calU16, calU17, and calU19, respectively. Amplification was performed using the Advantage GC2 polymerase enzyme (Clontech) and the following PCR conditions: initial denaturation at 95 °C for 3 min; 25 cycles at 94 °C for 30 s, 65–62 °C for 30 s, 68 °C for 35 s; final extension temperature at 68 °C for 5 min. Each PCR product was gel purified using the QIAquick gel extraction kit (Qiagen). Restriction digestion with NdeI/HindIII (New Englands Biolabs, NEB) followed by ligation using T4 DNA ligase (NEB) into a linearized dephosphorylated pET28a (Novagen) yielded pSECalU16, pSECalU17, and pSECalU19 for calU16, calU17, and calU19, respectively. Each plasmid was transformed into NEB DH5α chemically competent cells (NEB). Plasmids were purified using QIAprep Minispin kit (Qiagen). Each plasmid was verified by sequencing and subsequently transformed into the expression host BL21 (DE3) competent cells (NEB) to yield pSECalU16-E. coli, pSECalU17-E. coli, and pSECalU19-E. coli, respectively. For structural studies, plasmid preparation, overproduction, and purification were conducted following standard protocols of the Northeast Structural Genomics Consortium (NESG) to produce a uniformly 15N/13C-enriched and 5% biosynthetically directed 13C (NC5)-labeled protein samples for NMR spectroscopy and selenomethionine (Se-Met) labeled samples for X-ray crystallography and details can be found in Supporting Information Methods. (60, 61) In brief, calU16 was PCR amplified from genomic DNA and cloned into NdeI/XhoI-digested pET15 expression vector (NESG Clone ID MiR12–15.1; PSI:Biology Materials Repository clone ID MeCD000597015, see http://psimr.asu.edu/) to encode for the corresponding N-terminally tagged (MGHHHHHHSHM) fusion protein. The plasmid was transformed into codon-enhanced E. coli BL21 (DE3) pMGK cells for protein production. Additional plasmids and protocols employed for protein overproduction and purification for biochemical studies are described in the Supporting Information Methods.

Structure Determination of CalU16 by X-ray Crystallography

Initial crystallization conditions for Se-Met labeled CalU16 were identified at the Hauptmann-Woodward Institute high-throughput screening facility (62) and further optimized manually by microbatch methods at 18 °C. The protein was mixed in a 1:1 ratio with the precipitant containing 2.9 M sodium malonate pH 6.5. The crystals were cryo-protected by 10% (v/v) ethylene glycol and 2.6 M sodium malonate pH 6.5 prior to flash freezing in liquid nitrogen for data collection at 100 K. A single crystal of Se-Met labeled protein was used for data collection at a wavelength of 0.978 Å and diffracted to 2.5 Å resolution. The data was collected at beamline X4A at the National Synchrotron Light Source at Brookhaven National Laboratory, and further processed and scaled using HKL-2000 (Table 1). (63) Eight of the ten selenium sites in the asymmetric unit were successfully located by the SHELXD (64) program for initial phasing. RESOLVE (65) was utilized for automated model building and the model was further built by manual refitting with the program Coot. (66) The data were twinned, and a twin refinement was performed. The refinement involved iterations of manual model-building in Coot and Refmac. (67) The quality of the final structure was validated by PROCHECK. (68) The diffraction images illustrate diffuse scattering perpendicular to the 61 screw axis indicative of order/disorder (Supporting Information Figure S24). The effects of the disorder are also evident in the data anisotropy where the B-factor component along the 61 axis is less than half of the B-factor in the perpendicular direction. The unmodeled disorder leads to higher than expected R-factors and is likely contributing to the lower percentage of residues in the Ramachandran favored regions (Table 1). The atomic coordinates and structure factors have been deposited in the PDB (4FPW).

NMR Spectroscopy and Structure Determination of CalU16

NMR data were collected at 25 °C on [U-13C, 15N]- and U-15N, 5% biosynthetically directed 13C (NC5)-labeled samples in 300 μL buffered solution (0.9 mM CalU16, 0.02% NaN3, 10 mM DTT, 5 mM CaCl2, 100 mM NaCl, 1× proteinase inhibitors, 20 mM MES pH 6.5) in 5 mm Shigemi NMR tubes on a 600 MHz Varian Inova spectrometer with a 5 mm HCN cold probe and a 850 MHz Bruker Avance III NMR spectrometer equipped with a conventional 5 mm HCN probe. A description of NMR experiments and methods for structure determination and refinement can be found in Supporting Information Methods. The protein was monomeric under the conditions used in the NMR experiments based on analytical static light scattering in-line with gel filtration chromatography and correlation time estimates based on one-dimensional 15N T1 and T2 relaxation data (estimated τc 12.1 ns, Supporting Information Figure S1). The assigned 1H–15N HSQC spectrum is provided as Supporting Information Figure S2. Chemical shifts, NOESY peak lists, and raw FIDS were deposited in the BioMagResDB (BMRB accession number 18547). The final ensemble of 20 models and NMR resonance assignments were deposited to the Protein Data Bank (PDB: 2LUZ).

Resistance Assays

In vivo resistance assays were performed using overnight cultures of the tested strain diluted to OD600 ∼ 0.1 in molten LB agar supplemented with final concentration of 30 μg mL–1 kanamycin and 100 μM of isopropyl-β-d-thiogalactopyranoside (IPTG). The mixture was poured into Petri dishes and allowed to solidify. A stock solution of 1 was prepared in methanol at a concentration of 1 mg mL–1 and serial dilutions were prepared such that the final concentrations tested ranged from 0.1 to 10 μg μL–1. These were applied to sterile discs such that the final quantities of 1 on the discs were 1 ng, 10 ng, 50 ng, 100 ng, 1 μg, and 10 μg. The disks were allowed to air-dry, and applied aseptically to the plates. All plates were incubated at 37 °C for 14 h.

Protein Cleavage Assay

Final concentration of 1 μM of each protein in 50 mM Tris pH 8.0 in the presence of 1 mol equiv of 1 with and without 0.2 mM DTT in a final volume of 500 μL was allowed to incubate at 30 °C for 30 min. Reactions were lyophilized and resuspended in 25 μL of 50 mM Tris, pH 8.0. Cleavage was analyzed by mixing with equal volume of 2× sample buffer. A volume of 30 μL was analyzed by NuPAGE 4–12% Bis-Tris Gels (Life technologies) using protein ladder (Bio-Rad) as size indicator.

Trypsin Digestion and Mass Analysis

A total of 4 μM of each of CalU16 and CalU19 were added to 2 mol equiv of 1 in the presence of 0.2 mM DTT. Reactions were incubated at 30 °C for 30 min. A fraction of each reaction was subjected to SDS-PAGE to confirm completion of the reaction. For proteolytic digestion, the protein bands were excised from SDS-PAGE gel, washed with 50 mM ammonium bicarbonate, reduced with DTT, alkylated with iodoacetamide (IAA), and subjected to trypsin (for CalU16) or chymotrypsin (for CalU19) digestion at 37 °C for 16 h. Peptides resulting from the proteolytic digestion were extracted from gel pieces and analyzed by LC-MS/MS as previously described. (69) Briefly, the information-dependent acquisition of LC-MS/MS data were acquired using an LTQ Velos Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) coupled with a Nano-LC Ultra/cHiPLC-nanoflex HPLC system (Eksigent, Dublin, CA) through a nano-electrospray ionization source. (70) The results were subjected using ProteomeDiscoverer 1.3 software (Thermo Fisher Scientific, Waltham, MA) and MASCOT server. The cleavage sites were deduced from nonenzymatic peptides ending with an amine group (−NH2) from the Gly residue attached to a nontrypsin or nonchymotrypsin digestion site.

Determination of Minimum Inhibitory Concentrations (MICs)

Fourteen strains created in this study (Supporting Information Table S4, strains 2–15) were grown overnight in LB supplemented with kanamycin 30 mg L–1. Each overnight culture was diluted to OD600 ∼ 0.1 using an Overnight Express Autoinduction System 1 (Novagen) supplemented with kanamycin 30 mg L–1. Enediyne 1 was serially diluted 1:2 in DMSO such that the final concentrations ranged from 2.4 μM to 4.5 nM for pSE28a-E. coli and from 12 μM to 22 nM for all other strains. Ampicillin and kanamycin were used as positive and negative controls, respectively. All plates were incubated for 18 h at 37 °C at 250 rpm. The residual metabolic activity was monitored based on the irreversible reduction of resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide, blue) to resorufin (7-hydroxy-3H-phenoxazin-3-one, pink). For this assay, 10 μL of resazurin (final concentration 100 μM) was added to each well and allowed to incubate at 37 °C with shaking at 250 rpm for 1 h to allow viable cells to convert resazurin to resorufin. The well with the minimum concentration of 1 that did not display bacterial growth but did display a red color after incubation with resazurin was recorded as the MIC of that strain (Supporting Information Figure S9; Table 3).

Supporting Information

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Additional protocols and methods, supplemental figures, and supplemental tables. This material is available free of charge via the Internet at http://pubs.acs.org.

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Author Information

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  • Corresponding Authors
    • George N. Phillips - Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, United States;  Email: [email protected]
    • Jon S. Thorson - Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States;  Center for Pharmaceutical Research and Innovation (CPRI), College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States;  Email: [email protected]
  • Authors
    • Sherif I. Elshahawi - Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States;  Center for Pharmaceutical Research and Innovation (CPRI), College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States
    • Theresa A. Ramelot - Department of Chemistry and Biochemistry, Northeast Structural Genomics Consortium, Miami University, Oxford, Ohio 45056, United States
    • Jayaraman Seetharaman - Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, New York 10027, United States
    • Jing Chen - Department of Molecular and Cellular Biochemistry & Center for Structural Biology, College of Medicine, University of Kentucky, Lexington, Kentucky 40536, United States
    • Shanteri Singh - Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States;  Center for Pharmaceutical Research and Innovation (CPRI), College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States
    • Yunhuang Yang - Department of Chemistry and Biochemistry, Northeast Structural Genomics Consortium, Miami University, Oxford, Ohio 45056, United States
    • Kari Pederson - Complex Carbohydrate Research Center, Northeast Structural Genomics Consortium, University of Georgia, Athens, Georgia 30602, United States
    • Madan K. Kharel - Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States;  Center for Pharmaceutical Research and Innovation (CPRI), College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States
    • Rong Xiao - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, United States
    • Scott Lew - Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, New York 10027, United States
    • Ragothaman M. Yennamalli - Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, United States
    • Mitchell D. Miller - Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, United States
    • Fengbin Wang - Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, United States
    • Liang Tong - Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, New York 10027, United States
    • Gaetano T. Montelione - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, United States;  Department of Biochemistry, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, United States
    • Michael A. Kennedy - Department of Chemistry and Biochemistry, Northeast Structural Genomics Consortium, Miami University, Oxford, Ohio 45056, United States
    • Craig A. Bingman - Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States
    • Haining Zhu - Department of Molecular and Cellular Biochemistry & Center for Structural Biology, College of Medicine, University of Kentucky, Lexington, Kentucky 40536, United States
  • Notes
    The authors declare the following competing financial interest(s): J.S.T. is a co-founder of Centrose (Madison, WI).

Acknowledgment

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This work was supported by the National Institutes of Health (NIH) grants CA84374 (to J.S.T.), U01GM098248 (to G.N.P.), the National Center for Advancing Translational Sciences (UL1TR000117), a NIH PSI grant (U54-GM094597 to M.A.K. and G.T.M.) and the BioXFEL Science and Technology Center under National Science Foundation Grant No. 1231306. Enediynes for this study were generously provided by Pfizer (calicheamicin) and Bristol-Myers-Squibb (esperamicin and dynemicin). Proteomics analyses were conducted by the University of Kentucky Proteomics Core that is partially supported by grants from the National Institute of General Medical Sciences (P20GM103486), the National Cancer Institute (P30CA177558) and equipment acquired via a grant from National Center for Research Resources (1S10RR029127 to H.Z.). Protein NMR data collection was conducted at the Ohio Biomedicine Center of Excellence in Structural Biology and Metabolomics (Miami University). We thank Dr. J. Prestgard, Dr. T. Acton, and Dr. J. Everett at the Northeast Structural Genomics Consortium for technical assistance and J. Schwanof and R. Abramowitz for data collection assistance at beamline X4A, NSLS.

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    Biggins, J. B., Prudent, J. R., Marshall, D. J., and Thorson, J. S. (2006) A continuous assay for DNA cleavage using molecular break lights Methods Mol. Biol. 335, 83– 92
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    13
    A continuous assay for DNA cleavage using molecular break lights
    Biggins, John B.; Prudent, James R.; Marshall, David J.; Thorson, Jon S.
    Methods in Molecular Biology (Totowa, NJ, United States) (2006), 335 (Fluorescent Energy Transfer Nucleic Acid Probes), 83-92CODEN: MMBIED; ISSN:1064-3745. (Humana Press Inc.)
    Exploring the properties of mols. that cleave DNA (i.e., enzymic nucleases, chem. footprinting agents, and naturally occurring DNA cleaving antibiotics) has been an ongoing process with benefits extending toward both lab. and clin. applications. Despite the progress that has been made toward understanding the mechanics of DNA cleavage, a simple and continuous assay for detecting DNA cleavage has been lacking. Herein, the authors describe the mol. break light assay, wherein a single oligonucleotide modified by a 5'-fluorophore-3'-quencher pair adopting a stem-loop structure with an appropriate DNA recognition site, provides for the rapid assaying of DNA cleavage with high sensitivity. Furthermore, the described methodol. is highly convenient in that it is readily adaptable to common lab. fluorometers and multi-well plate/array systems, which may provide the basis for high-throughput screening of novel DNA cleaving agents. This assay may also be further extended to natural or "unnatural" transcription factor protection assay systems.
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  14. 14
    Danishefsky, S. J. and Shair, M. D. (1996) Observations in the chemistry and biology of cyclic enediyne antibiotics: Total syntheses of calicheamicin γ1I and dynemicin A J. Org. Chem. 61, 16– 44
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    Nicolaou, K. C., Hale, C. R. H., and Nilewski, C. (2012) A total synthesis trilogy: Calicheamicin γ1(I), Taxol, and brevetoxin A Chem. Rec. 12, 407– 441
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    A total synthesis trilogy: Calicheamicin γ1I, Taxol and brevetoxin A
    Nicolaou, K. C.; Hale, Christopher R. H.; Nilewski, Christian
    Chemical Record (2012), 12 (4), 407-441CODEN: CRHEAK; ISSN:1527-8999. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Detailed behind-the-scenes accounts of the total syntheses of calicheamicin γ1I, Taxol and brevetoxin A are discussed with particular emphasis placed on strategies and tactics employed in these campaigns. DOI 10.1002/tcr.201200005.
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  16. 16
    Ahlert, J., Shepard, E., Lomovskaya, N., Zazopoulos, E., Staffa, A., Bachmann, B. O., Huang, K., Fonstein, L., Czisny, A., Whitwam, R. E., Farnet, C. M., and Thorson, J. S. (2002) The calicheamicin gene cluster and its iterative type I enediyne PKS Science 297, 1173– 1176
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    16
    The calicheamicin gene cluster and its iterative type I enediyne PKS
    Ahlert, Joachim; Shepard, Erica; Lomovskaya, Natalia; Zazopoulos, Emmanuel; Staffa, Alfredo; Bachmann, Brian O.; Huang, Kexue; Fonstein, Leonid; Czisny, Anne; Whitwam, Ross E.; Farnet, Chris M.; Thorson, Jon S.
    Science (Washington, DC, United States) (2002), 297 (5584), 1173-1176CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    The enediynes exemplify nature's ingenuity. We have cloned and characterized the biosynthetic locus coding for perhaps the most notorious member of the nonchromoprotein enediyne family, calicheamicin. This gene cluster contains an unusual polyketide synthase (PKS) that is demonstrated to be essential for enediyne biosynthesis. Comparison of the calicheamicin locus with the locus encoding the chromoprotein enediyne C-1027 reveals that the enediyne PKS is highly conserved among these distinct enediyne families. Contrary to previous hypotheses, this suggests that the chromoprotein and nonchromoprotein enediynes are generated by similar biosynthetic pathways.
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    Liu, W., Ahlert, J., Gao, Q., Wendt-Pienkowski, E., Shen, B., and Thorson, J. S. (2003) Rapid PCR amplification of minimal enediyne polyketide synthase cassettes leads to a predictive familial classification model Proc. Natl. Acad. Sci. U.S.A. 100, 11959– 11963
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    17
    Rapid PCR amplification of minimal enediyne polyketide synthase cassettes leads to a predictive familial classification model
    Liu, Wen; Ahlert, Joachim; Gao, Qunjie; Wendt-Pienkowski, Evelyn; Shen, Ben; Thorson, Jon S.
    Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (21), 11959-11963CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    A universal PCR method for the rapid amplification of minimal enediyne polyketide synthase (PKS) genes and the application of this methodol. to clone remaining prototypical genes from producers of structurally detd. enediynes in both family types are presented. A phylogenetic anal. of the new pool of bona fide enediyne PKS genes, consisting of three from 9-membered producers (neocarzinostatin, C1027, and maduropeptin) and three from 10-membered producers (calicheamicin, dynemicin, and esperamicin), reveals a clear genotypic distinction between the two structural families from which to form a predictive model. The results from this study support the postulation that the minimal enediyne PKS helps define the structural divergence of the enediyne core and provides the key tools for generating enediyne hybrid genes/mol. scaffolds; by using the model, a classification is also provided for the unknown enediyne PKS genes previously identified via genome scanning.
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  18. 18
    Biggins, J. B., Onwueme, K. C., and Thorson, J. S. (2003) Resistance to enediyne antitumor antibiotics by CalC self-sacrifice Science 301, 1537– 1541
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    Singh, S., Hager, M. H., Zhang, C., Griffith, B. R., Lee, M. S., Hallenga, K., Markley, J. L., and Thorson, J. S. (2006) Structural insight into the self-sacrifice mechanism of enediyne resistance ACS Chem. Biol. 1, 451– 460
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    19
    Structural insight into the self-sacrifice mechanism of enediyne resistance
    Singh, Shanteri; Hager, Martin H.; Zhang, Changsheng; Griffith, Byron R.; Lee, Min S.; Hallenga, Klaas; Markley, John L.; Thorson, Jon S.
    ACS Chemical Biology (2006), 1 (7), 451-460CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    The recent discovery of the first "self-sacrifice" mechanism for bacterial resistance to the enediyne antitumor antibiotics, where enediyne-induced proteolysis of the resistance protein CalC inactivates both the highly reactive metabolite and the resistance protein, revealed yet another ingenious bacterial mechanism for controlling reactive metabolites. As reported herein, the first 3D structures of CalC and CalC in complex with calicheamicin (CLM) divulge CalC to be a member of the steroidogenic acute regulatory protein (StAR)-related transfer (START) domain superfamily. In contrast to previous studies of proteins known to bind DNA-damaging natural products (e.g., bleomycins, mitomycins, and 9-membered chromoprotein enediynes), this is the 1st demonstrated involvement of a START domain fold. Consistent with the CalC self-sacrifice mechanism, CLM in complex with CalC is positioned for direct H abstraction from Gly113 to initiate the oxidative proteolysis-based resistance mechanism. These structural studies also illuminate, for the 1st time, a small DNA-binding region within CalC that may serve to localize CalC to the enediyne target (DNA). Given the role of START domains in nuclear/cytosolic transport and translocation, this structural study also may implicate START domains as post-endocytotic intracellular chaperones for enediyne-based therapeutics such as MyloTarg.
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  20. 20
    Zhang, C., Griffith, B. R., Fu, Q., Albermann, C., Fu, X., Lee, I.-K., Li, L., and Thorson, J. S. (2006) Exploiting the reversibility of natural product glycosyltransferase-catalyzed reactions Science 313, 1291– 1294
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    20
    Exploiting the Reversibility of Natural Product Glycosyltransferase-Catalyzed Reactions
    Zhang, Changsheng; Griffith, Byron R.; Fu, Qiang; Albermann, Christoph; Fu, Xun; Lee, In-Kyoung; Li, Lingjun; Thorson, Jon S.
    Science (Washington, DC, United States) (2006), 313 (5791), 1291-1294CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Glycosyltransferases (GTs), an essential class of ubiquitous enzymes, are generally perceived as unidirectional catalysts. In contrast, we report that four glycosyltransferases from two distinct natural product biosynthetic pathways-calicheamicin and vancomycin-readily catalyze reversible reactions, allowing sugars and aglycons to be exchanged with ease. As proof of the broader applicability of these new reactions, more than 70 differentially glycosylated calicheamicin and vancomycin variants are reported. This study suggests the reversibility of GT-catalyzed reactions may be general and useful for generating exotic nucleotide sugars, establishing in vitro GT activity in complex systems, and enhancing natural product diversity.
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  21. 21
    Johnson, H. D. and Thorson, J. S. (2008) Characterization of CalE10, the N-oxidase involved in calicheamicin hydroxyaminosugar formation J. Am. Chem. Soc. 130, 17662– 17663
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    Belecki, K., Crawford, J. M., and Townsend, C. A. (2009) Production of octaketide polyenes by the calicheamicin polyketide synthase CalE8: Implications for the biosynthesis of enediyne core structures J. Am. Chem. Soc. 131, 12564– 12566
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    22
    Production of Octaketide Polyenes by the Calicheamicin Polyketide Synthase CalE8: Implications for the Biosynthesis of Enediyne Core Structures
    Belecki, Katherine; Crawford, Jason M.; Townsend, Craig A.
    Journal of the American Chemical Society (2009), 131 (35), 12564-12566CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Enediyne antibiotics are categorized according to the presence of either a 9- or 10-membered ring within their polyketide-derived core structures. Recent literature reports have favored the notion that biosynthetic divergence of the two structural families is detd. by the enediyne polyketide synthases (PKSs) alone. We now disclose the simultaneous in vitro prodn. of three octaketide polyenes by biosynthetic enzymes for the 10-membered enediyne calicheamicin γ1I, including the elusive β-keto acid precursor to a previously described C15 Me hexaenone. Alongside these two polyene products, we have addnl. detected a hydrocarbon heptaene previously isolated only from 9-membered enediyne systems. The discovery of the heptaene in the calicheamicin system promotes a more convergent model for the early steps of enediyne biosynthesis. Furthermore, the synthesis of this set of octaketides by the enediyne PKS CalE8 and thioesterase CalE7 suggests, in contrast to recent biosynthetic proposals, that accessory enzymes may be necessary to initiate differentiation to 9- or 10-membered enediyne precursors, either by modulation of enediyne PKS activity or by interception and modification of polyketide chain-extension intermediates.
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  23. 23
    Horsman, G. P., Chen, Y., Thorson, J. S., and Shen, B. (2010) Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes Proc. Natl. Acad. Sci. U.S.A. 107, 11331– 11335
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    Gantt, R. W., Peltier-Pain, P., Singh, S., Zhou, M., and Thorson, J. S. (2013) Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis Proc. Natl. Acad. Sci. U.S.A. 110, 7648– 7653
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    24
    Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis
    Gantt, Richard W.; Peltier-Pain, Pauline; Singh, Shanteri; Zhou, Maoquan; Thorson, Jon S.
    Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (19), 7648-7653, S7648/1-S7648/72CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed max. improvements in kcat/Km of >400-fold and >15-fold for formation of NDP-glucoses and UDP-sugars, resp. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP-sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.
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    Belecki, K. and Townsend, C. A. (2012) Environmental control of the calicheamicin polyketide synthase leads to detection of a programmed octaketide and a proposal for enediyne biosynthesis Angew. Chem., Int. Ed. 51, 11316– 11319
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    Belecki, K. and Townsend, C. A. (2013) Biochemical determination of enzyme-bound metabolites: Preferential accumulation of a programmed octaketide on the enediyne polyketide synthase CalE8 J. Am. Chem. Soc. 135, 14339– 14348
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    Ricart, A. D. (2011) Antibody-drug conjugates of calicheamicin derivative: Gemtuzumab Ozogamicin and Inotuzumab Ozogamicin Clin. Cancer Res. 17, 6417– 6427
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    Antibody-Drug Conjugates of Calicheamicin Derivative: Gemtuzumab Ozogamicin and Inotuzumab Ozogamicin
    Ricart, Alejandro D.
    Clinical Cancer Research (2011), 17 (20), 6417-6427CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
    A review. Antibody-drug conjugates (ADC) are an attractive approach for the treatment of acute myeloid leukemia and non-Hodgkin lymphomas, which in most cases, are inherently sensitive to cytotoxic agents. CD33 and CD22 are specific markers of myeloid leukemias and B-cell malignancies, resp. These endocytic receptors are ideal for an ADC strategy because they can effectively carry the cytotoxic payload into the cell. Gemtuzumab ozogamicin (GO, Mylotarg) and inotuzumab ozogamicin consist of a deriv. of calicheamicin (a potent DNA-binding cytotoxic antibiotic) linked to a humanized monoclonal IgG4 antibody directed against CD33 or CD22, resp. Both of these ADCs have a target-mediated pharmacokinetic disposition. GO was the first drug to prove the ADC concept in the clinic, specifically in phase II studies that included substantial proportions of older patients with relapsed acute myeloid leukemia. In contrast, in phase III studies, it has thus far failed to show clin. benefit in first-line treatment in combination with std. chemotherapy. Inotuzumab ozogamicin has shown remarkable clin. activity in relapsed/refractory B-cell non-Hodgkin lymphoma, and it has started phase III evaluation. The safety profile of these ADCs includes reversible myelosuppression (esp. neutropenia and thrombocytopenia), elevated hepatic transaminases, and hyperbilirubinemia. There have been postmarketing reports of hepatotoxicity, esp. veno-occlusive disease, assocd. with GO. The incidence is ∼2%, but patients who undergo hematopoietic stem cell transplantation have an increased risk. As we steadily move toward the goal of personalized medicine, these kinds of agents will provide a unique opportunity to treat selected patient subpopulations based on the expression of their specific tumor targets. Clin Cancer Res; 17(20); 6417-27.
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    Trail, P. A. (2013) Antibody drug conjugates as cancer therapeutics Antibodies 2, 113– 129
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    Krissinel, E. and Henrick, K. (2007) Inference of macromolecular assemblies from crystalline state J. Mol. Biol. 372, 774– 797
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    29
    Inference of Macromolecular Assemblies from Crystalline State
    Krissinel, Evgeny; Henrick, Kim
    Journal of Molecular Biology (2007), 372 (3), 774-797CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    The authors discuss basic phys.-chem. principles underlying the formation of stable macromol. complexes, which in many cases are likely to be the biol. units performing a certain physiol. function. The authors also consider available theor. approaches to the calcn. of macromol. affinity and entropy of complexation. The latter is shown to play an important role and make a major effect on complex size and symmetry. The authors develop a new method, based on chem. thermodn., for automatic detection of macromol. assemblies in the Protein Data Bank (PDB) entries that are the results of x-ray diffraction expts. As found, biol. units may be recovered at 80-90% success rate, which makes x-ray crystallog. an important source of exptl. data on macromol. complexes and protein-protein interactions. The method is implemented as a public WWW service (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html).
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    Iyer, L. M., Koonin, E. V., and Aravind, L. (2001) Adaptations of the helix-grip fold for ligand binding and catalysis in the START domain superfamily Proteins 43, 134– 144
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    Thorsell, A.-G., Lee, W. H., Persson, C., Siponen, M. I., Nilsson, M., Busam, R. D., Kotenyova, T., Schüler, H., and Lehtiö, L. (2011) Comparative structural analysis of lipid binding START domains PloS One 6, e19521
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    Alpy, F. and Tomasetto, C. (2005) Give lipids a START: The StAR-related lipid transfer (START) domain in mammals J. Cell Sci. 118, 2791– 2801
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    Give lipids a START: The StAR-related lipid transfer (START) domain in mammals
    Alpy, Fabien; Tomasetto, Catherine
    Journal of Cell Science (2005), 118 (13), 2791-2801CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)
    A review. The steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain is a protein module of ∼210 residues that binds lipids, including sterols. Fifteen mammalian proteins, STARD1-STARD15, possess a START domain and these can be grouped into 6 subfamilies. Cholesterol, 25-hydroxycholesterol, phosphatidylcholine, phosphatidylethanolamine, and ceramides are ligands for STARD1/STARD3/STARD5, STARD5, STARD2/STARD10, STARD10 and STARD11, resp. The lipids or sterols bound by the remaining 9 START proteins are unknown. Recent studies have shown that the C-terminal end of the domain plays a fundamental role, forming a lid over a deep lipid-binding pocket that shields the ligand from the external environment. The START domain can be regarded as a lipid-exchange and/or a lipid-sensing domain. Mammalian START proteins have diverse expression patterns and can be found free in the cytoplasm, attached to membranes or in the nucleus. They appear to function in a variety of distinct physiol. processes, such as lipid transfer between intracellular compartments, lipid metab., and modulation of signaling events. Mutation or misexpression of START proteins is linked to pathol. processes, including genetic disorders, autoimmune disease, and cancer.
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    Holm, L. and Rosenström, P. (2010) Dali server: Conservation mapping in 3D Nucleic Acids Res. 38, W545– 549
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    Dali server: conservation mapping in 3D
    Holm, Liisa; Rosenstrom, Paivi
    Nucleic Acids Research (2010), 38 (Web Server), W545-W549CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Our web site (http://ekhidna.biocenter.helsinki.fi/daliserver) runs the Dali program for protein structure comparison. The web site consists of three parts: (i) the Dali server compares newly solved structures against structures in the Protein Data Bank (PDB), (ii) the Dali database allows browsing precomputed structural neighborhoods and (iii) the pairwise comparison generates suboptimal alignments for a pair of structures. Each part has its own query form and a common format for the results page. The inputs are either PDB identifiers or novel structures uploaded by the user. The results pages are hyperlinked to aid interactive anal. The web interface is simple and easy to use. The key purpose of interactive anal. is to check whether conserved residues line up in multiple structural alignments and how conserved residues and ligands cluster together in multiple structure superimpositions. In favorable cases, protein structure comparison can lead to evolutionary discoveries not detected by sequence anal.
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  34. 34
    Krissinel, E. and Henrick, K. (2004) Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2256– 2268
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    Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions
    Krissinel, E.; Henrick, K.
    Acta Crystallographica, Section D: Biological Crystallography (2004), D60 (12, Pt. 1), 2256-2268CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
    The present paper describes the SSM algorithm of protein structure comparison in three dimensions, which includes an original procedure of matching graphs built on the protein's secondary-structure elements, followed by an iterative three-dimensional alignment of protein backbone Cα atoms. The SSM results are compared with those obtained from other protein comparison servers, and the advantages and disadvantages of different scores that are used for structure recognition are discussed. A new score, balancing the r.m.s.d. and alignment length Nalign, is proposed. It is found that different servers agree reasonably well on the new score, while showing considerable differences in r.m.s.d. and Nalign.
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    Ames, B. D., Korman, T. P., Zhang, W., Smith, P., Vu, T., Tang, Y., and Tsai, S.-C. (2008) Crystal structure and functional analysis of tetracenomycin ARO/CYC: Implications for cyclization specificity of aromatic polyketides Proc. Natl. Acad. Sci. U.S.A. 105, 5349– 5354
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    Michalska, K., Fernandes, H., Sikorski, M., and Jaskolski, M. (2010) Crystal structure of Hyp-1, a St. John’s Wort protein implicated in the biosynthesis of hypericin J. Struct. Biol. 169, 161– 171
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    Ilari, A., Franceschini, S., Bonamore, A., Arenghi, F., Botta, B., Macone, A., Pasquo, A., Bellucci, L., and Boffi, A. (2009) Structural basis of enzymatic (S)-norcoclaurine biosynthesis J. Biol. Chem. 284, 897– 904
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    Structural basis of enzymatic (S)-norcoclaurine biosynthesis
    Ilari, Andrea; Franceschini, Stefano; Bonamore, Alessandra; Arenghi, Fabio; Botta, Bruno; Macone, Alberto; Pasquo, Alessandra; Bellucci, Luca; Boffi, Alberto
    Journal of Biological Chemistry (2009), 284 (2), 897-904CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Norcoclaurine synthase (NCS) catalyzes the stereospecific Pictet-Spengler cyclization between dopamine and 4-hydroxyphenylacetaldehyde, the key step in the benzylisoquinoline alkaloid biosynthetic pathway. Here, the crystallog. structure of NCS from Thalictrum flavum in its complex with its substrate, dopamine, and the nonreactive substrate analog, 4-hydroxybenzaldehyde, was solved at 2.1 Å resoln. NCS shared no common features with the functionally correlated "Pictet-Spenglerases" that catalyze the 1st step of the indole alkaloid pathways and conformed to the overall fold of the Bet v1-like protein. The active site of NCS was located within a 20-Å-long catalytic tunnel and was shaped by the side-chains of the Tyr-108, Lys-122, Asp-141, and Glu-110 residues. The geometry of the amino acid side-chains with respect to the substrates revealed the structural determinants that govern the mechanism of the stereoselective Pictet-Spengler cyclization, thus establishing an excellent foundation for the understanding of the finer details of the catalytic process. Site-directed mutations of the relevant residues confirmed the assignment based on the crystallog. findings.
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    Li, W., Wang, L., Sheng, X., Yan, C., Zhou, R., Hang, J., Yin, P., and Yan, N. (2013) Molecular basis for the selective and ABA-independent inhibition of PP2CA by PYL13 Cell Res. 23, 1369– 1379
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    Cell Research (2013), 23 (12), 1369-1379CODEN: CREEB6; ISSN:1001-0602. (NPG Nature Asia-Pacific)
    PYR1/PYL/RCAR family proteins (PYLs) are well-characterized abscisic acid (ABA) receptors. Among the 14 PYL members in Arabidopsis thaliana, PYL13 is ABA irresponsive and its function has remained elusive. Here, we show that PYL13 selectively inhibits the phosphatase activity of PP2CA independent of ABA. The crystal structure of PYL13-PP2CA complex, which was detd. at 2.4 Å resoln., elucidates the mol. basis for the specific recognition between PP2CA and PYL13. In addn. to the canonical interactions between PYLs and PP2Cs, an extra interface is identified involving an element in the vicinity of a previously uncharacterized CCCH zinc-finger (ZF) motif in PP2CA. Sequence blast identified another 56 ZF-contg. PP2Cs, all of which are from plants. The structure also reveals the mol. determinants for the ABA irresponsiveness of PYL13. Finally, biochem. anal. suggests that PYL13 may hetero-oligomerize with PYL10. These two PYLs antagonize each other in their resp. ABA-independent inhibitions of PP2Cs. The biochem. and structural studies provide important insights into the function of PYL13 in the stress response of plant and set up a foundation for future biotechnol. applications of PYL13.
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    The mammalian START domain protein family in lipid transport in health and disease
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    Journal of Endocrinology (2012), 212 (3), 257-275CODEN: JOENAK; ISSN:0022-0795. (BioScientifica Ltd.)
    A review. Lipid transfer proteins of the steroidogenic acute regulatory protein-related lipid transfer (START) domain family are defined by the presence of a conserved ∼210 amino acid sequence that folds into an α/β helix-grip structure forming a hydrophobic pocket for ligand binding. The mammalian START proteins bind diverse ligands, such as cholesterol, oxysterols, phospholipids, sphingolipids, and possibly fatty acids, and have putative roles in non-vesicular lipid transport, thioesterase enzymic activity, and tumor suppression. However, the biol. functions of many members of the START domain protein family are not well established. Recent research has focused on characterizing the cell-type distribution and regulation of the START proteins, examg. the specificity and directionality of lipid transport, and identifying disease states assocd. with dysregulation of START protein expression. This review summarizes the current concepts of the proposed physiol. and pathol. roles for the mammalian START domain proteins in cholesterol and lipid trafficking.
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    Whisstock, James C.; Lesk, Arthur M.
    Quarterly Reviews of Biophysics (2003), 36 (3), 307-340CODEN: QURBAW; ISSN:0033-5835. (Cambridge University Press)
    A review. The sequence of a genome contains the plans of the possible life of an organism, but implementation of genetic information depends on the functions of the proteins and nucleic acids that it encodes. Many individual proteins of known sequence and structure present challenges to the understanding of their function. In particular, a no. of genes responsible for diseases have been identified but their specific functions are unknown. Whole-genome sequencing projects are a major source of proteins of unknown function. Annotation of a genome involves assignment of functions to gene products, in most cases on the basis of amino-acid sequence alone. 3D structure can aid the assignment of function, motivating the challenge of structural genomics projects to make structural information available for novel uncharacterized proteins. Structure-based identification of homologs often succeeds where sequence-alone-based methods fail, because in many cases evolution retains the folding pattern long after sequence similarity becomes undetectable. Nevertheless, prediction of protein function from sequence and structure is a difficult problem, because homologous proteins often have different functions. Many methods of function prediction rely on identifying similarity in sequence and/or structure between a protein of unknown function and one or more well-understood proteins. Alternative methods include inferring conservation patterns in members of a functionally uncharacterized family for which many sequences and structures are known. However, these inferences are tenuous. Such methods provide reasonable guesses at function, but are far from foolproof. It is therefore fortunate that the development of whole-organism approaches and comparative genomics permits other approaches to function prediction when the data are available. These include the use of protein-protein interaction patterns, and correlations between occurrences of related proteins in different organisms, as indicators of functional properties. Even if it is possible to ascribe a particular function to a gene product, the protein may have multiple functions. A fundamental problem is that function is in many cases an ill-defined concept. In this article we review the state of the art in function prediction and describe some of the underlying difficulties and successes.
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    Schnoes, A. M., Brown, S. D., Dodevski, I., and Babbitt, P. C. (2009) Annotation error in public databases: Misannotation of molecular function in enzyme superfamilies PLoS Comput. Biol. 5, e1000605
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    Schnoes Alexandra M; Brown Shoshana D; Dodevski Igor; Babbitt Patricia C
    PLoS computational biology (2009), 5 (12), e1000605 ISSN:.
    Due to the rapid release of new data from genome sequencing projects, the majority of protein sequences in public databases have not been experimentally characterized; rather, sequences are annotated using computational analysis. The level of misannotation and the types of misannotation in large public databases are currently unknown and have not been analyzed in depth. We have investigated the misannotation levels for molecular function in four public protein sequence databases (UniProtKB/Swiss-Prot, GenBank NR, UniProtKB/TrEMBL, and KEGG) for a model set of 37 enzyme families for which extensive experimental information is available. The manually curated database Swiss-Prot shows the lowest annotation error levels (close to 0% for most families); the two other protein sequence databases (GenBank NR and TrEMBL) and the protein sequences in the KEGG pathways database exhibit similar and surprisingly high levels of misannotation that average 5%-63% across the six superfamilies studied. For 10 of the 37 families examined, the level of misannotation in one or more of these databases is >80%. Examination of the NR database over time shows that misannotation has increased from 1993 to 2005. The types of misannotation that were found fall into several categories, most associated with "overprediction" of molecular function. These results suggest that misannotation in enzyme superfamilies containing multiple families that catalyze different reactions is a larger problem than has been recognized. Strategies are suggested for addressing some of the systematic problems contributing to these high levels of misannotation.
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    Baker, David; Sali, Andrej
    Science (Washington, DC, United States) (2001), 294 (5540), 93-96CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Genome sequencing projects are producing linear amino acid sequences, but full understanding of the biol. role of these proteins will require knowledge of their structure and function. Although exptl. structure detn. methods are providing high-resoln. structure information about a subset of the proteins, computational structure prediction methods will provide valuable information for the large fraction of sequences whose structures will not be detd. exptl. The first class of protein structure prediction methods, including threading and comparative modeling, rely on detectable similarity spanning most of the modeled sequence and at least one known structure. The second class of methods, de novo or ab initio methods, predict the structure from sequence alone, without relying on similarity at the fold level between the modeled sequence and any of the known structures. In this Viewpoint, we begin by describing the essential features of the methods, the accuracy of the models, and their application to the prediction and understanding of protein function, both for single proteins and on the scale of whole genomes. We then discuss the important role that protein structure prediction methods play in the growing worldwide effort in structural genomics.
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    Hermann, J. C., Marti-Arbona, R., Fedorov, A. A., Fedorov, E., Almo, S. C., Shoichet, B. K., and Raushel, F. M. (2007) Structure-based activity prediction for an enzyme of unknown function Nature 448, 775– 779
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    Hermann, Johannes C.; Marti-Arbona, Ricardo; Fedorov, Alexander A.; Fedorov, Elena; Almo, Steven C.; Shoichet, Brian K.; Raushel, Frank M.
    Nature (London, United Kingdom) (2007), 448 (7155), 775-779CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    With many genomes sequenced, a pressing challenge in biol. is predicting the function of the proteins that the genes encode. When proteins are unrelated to others of known activity, bioinformatics inference for function becomes problematic. It would thus be useful to interrogate protein structures for function directly. Here, we predict the function of an enzyme of unknown activity, Tm0936 from Thermotoga maritima, by docking high-energy intermediate forms of thousands of candidate metabolites. The docking hit list was dominated by adenine analogs, which appeared to undergo C6-deamination. Four of these, including 5-methylthioadenosine and S-adenosylhomocysteine (SAH), were tested as substrates, and three had substantial catalytic rate consts. (105 M-1 s-1). The x-ray crystal structure of the complex between Tm0936 and the product resulting from the deamination of SAH, S-inosylhomocysteine, was detd., and it corresponded closely to the predicted structure. The deaminated products can be further metabolized by T. maritima in a previously uncharacterized SAH degrdn. pathway. Structure-based docking with high-energy forms of potential substrates may be a useful tool to annotate enzymes for function.
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    Nature Reviews Microbiology (2007), 5 (3), 175-186CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
    A review. Over the millennia, microorganisms have evolved evasion strategies to overcome a myriad of chem. and environmental challenges, including antimicrobial drugs. Even before the first clin. use of antibiotics more than 60 years ago, resistant organisms had been isolated. Moreover, the potential problem of the widespread distribution of antibiotic resistant bacteria was recognized by scientists and health-care specialists from the initial use of these drugs. Why is resistance inevitable and where does it come from. Understanding the mol. diversity that underlies resistance will inform our use of these drugs and guide efforts to develop new efficacious antibiotics.
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    Cundliffe, Eric; Demain, Arnold L.
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    A review. Many microbes synthesize potentially autotoxic antibiotics, mainly as secondary metabolites, against which they need to protect themselves. This is done in various ways, ranging from target-based strategies (i.e., modification of normal drug receptors or de novo synthesis of the latter in drug-resistant form) to the adoption of metabolic shielding and/or efflux strategies that prevent drug-target interactions. These self-defense mechanisms have been studied most intensively in antibiotic-producing prokaryotes, of which the most prolific are the actinomycetes. Only a few documented examples pertain to lower eukaryotes while higher organisms have hardly been addressed in this context. Thus, many plant alkaloids, variously described as herbivore repellents or nitrogen excretion devices, are truly antibiotics-even if toxic to humans. As just one example, bulbs of Narcissus spp. (including the King Alfred daffodil) accumulate narciclasine that binds to the larger subunit of the eukaryotic ribosome and inhibits peptide bond formation. However, ribosomes in the Amaryllidaceae have not been tested for possible resistance to narciclasine and other alkaloids. Clearly, the prevalence of suicide avoidance is likely to extend well beyond the remit of the present article.
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    Allan, C. M., Hill, S., Morvaridi, S., Saiki, R., Johnson, J. S., Liau, W.-S., Hirano, K., Kawashima, T., Ji, Z., Loo, J. A., Shepherd, J. N., and Clarke, C. F. (2013) A conserved START domain coenzyme Q-binding polypeptide is required for efficient Q biosynthesis, respiratory electron transport, and antioxidant function in Saccharomyces cerevisiae Biochim. Biophys. Acta 1831, 776– 791
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    Weigel, L. M., Clewell, D. B., Gill, S. R., Clark, N. C., McDougal, L. K., Flannagan, S. E., Kolonay, J. F., Shetty, J., Killgore, G. E., and Tenover, F. C. (2003) Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus Science 302, 1569– 1571
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    Weigel, Linda M.; Clewell, Don B.; Gill, Steven R.; Clark, Nancye C.; McDougal, Linda K.; Flannagan, Susan E.; Kolonay, James F.; Shetty, Jyoti; Killgore, George E.; Tenover, Fred C.
    Science (Washington, DC, United States) (2003), 302 (5650), 1569-1571CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Vancomycin is usually reserved for treatment of serious infections, including those caused by multidrug-resistant Staphylococcus aureus. A clin. isolate of S. aureus with high-level resistance to vancomycin (minimal inhibitory concn. = 1024 μg/mL) was isolated in June 2002. This isolate harbored a 57.9-kilobase multiresistance conjugative plasmid within which Tn1546 (vanA) was integrated. Addnl. elements on the plasmid encoded resistance to trimethoprim (dfrA), β-lactams (blaZ), aminoglycosides (aacA-aphD), and disinfectants (qacC). Genetic analyses suggest that the long-anticipated transfer of vancomycin resistance to a methicillin-resistant S. aureus occurred in vivo by interspecies transfer of Tn1546 from a co-isolate of Enterococcus faecalis.
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    Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P., and Drummond, A. (2012) Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data Bioinformatics 28, 1647– 1649
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    Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data
    Kearse Matthew; Moir Richard; Wilson Amy; Stones-Havas Steven; Cheung Matthew; Sturrock Shane; Buxton Simon; Cooper Alex; Markowitz Sidney; Duran Chris; Thierer Tobias; Ashton Bruce; Meintjes Peter; Drummond Alexei
    Bioinformatics (Oxford, England) (2012), 28 (12), 1647-9 ISSN:.
    UNLABELLED: The two main functions of bioinformatics are the organization and analysis of biological data using computational resources. Geneious Basic has been designed to be an easy-to-use and flexible desktop software application framework for the organization and analysis of biological data, with a focus on molecular sequences and related data types. It integrates numerous industry-standard discovery analysis tools, with interactive visualizations to generate publication-ready images. One key contribution to researchers in the life sciences is the Geneious public application programming interface (API) that affords the ability to leverage the existing framework of the Geneious Basic software platform for virtually unlimited extension and customization. The result is an increase in the speed and quality of development of computation tools for the life sciences, due to the functionality and graphical user interface available to the developer through the public API. Geneious Basic represents an ideal platform for the bioinformatics community to leverage existing components and to integrate their own specific requirements for the discovery, analysis and visualization of biological data. AVAILABILITY AND IMPLEMENTATION: Binaries and public API freely available for download at http://www.geneious.com/basic, implemented in Java and supported on Linux, Apple OSX and MS Windows. The software is also available from the Bio-Linux package repository at http://nebc.nerc.ac.uk/news/geneiousonbl.
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    Acton, T. B., Xiao, R., Anderson, S., Aramini, J., Buchwald, W. A., Ciccosanti, C., Conover, K., Everett, J., Hamilton, K., Huang, Y. J., Janjua, H., Kornhaber, G., Lau, J., Lee, D. Y., Liu, G., Maglaqui, M., Ma, L., Mao, L., Patel, D., Rossi, P., Sahdev, S., Shastry, R., Swapna, G. V. T., Tang, Y., Tong, S., Wang, D., Wang, H., Zhao, L., and Montelione, G. T. (2011) Preparation of protein samples for NMR structure, function, and small-molecule screening studies Methods Enzymol. 493, 21– 60
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    Preparation of protein samples for NMR structure, function, and small-molecule screening studies
    Acton, Thomas B.; Xiao, Rong; Anderson, Stephen; Aramini, James; Buchwald, William A.; Ciccosanti, Colleen; Conover, Ken; Everett, John; Hamilton, Keith; Huang, Yuanpeng Janet; Janjua, Haleema; Kornhaber, Gregory; Lau, Jessica; Lee, Dong Yup; Liu, Gaohua; Maglaqui, Melissa; Ma, Lichung; Mao, Lei; Patel, Dayaban; Rossi, Paolo; Sahdev, Seema; Shastry, Ritu; Swapna, G. V. T.; Tang, Yeufeng; Tong, Saichiu; Wang, Dongyan; Wang, Huang; Zhao, Li; Montelione, Gaetano T.
    Methods in Enzymology (2011), 493 (Fragment-Based Drug Design), 21-60CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)
    A review. In this chapter, we conc. on the prodn. of high-quality protein samples for NMR studies, in particular, we provide an in-depth description of recent advances in the prodn. of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein prodn. platform of the Northeast Structural Genomics Consortium and outline our high-throughput strategies for producing high-quality protein samples for NMR studies. Our strategy is based on the cloning, expression, and purifn. of 6 × -His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and anal. expression systems, parallel preparative scale fermn., and high-throughput purifn. protocols. The 6 × -His affinity tag allows for a similar two-step purifn. procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (>97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5000 of these proteins have been purified to homogeneity in tens of milligram quantities, resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this chapter describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purifn., and biophys. screening technologies are suitable for implementation in a large individual lab. or by a small group of collaborating investigators for structural biol., functional proteomics, ligand screening, and structural genomics research.
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    Xiao, R., Anderson, S., Aramini, J., Belote, R., Buchwald, W. A., Ciccosanti, C., Conover, K., Everett, J. K., Hamilton, K., Huang, Y. J., Janjua, H., Jiang, M., Kornhaber, G. J., Lee, D. Y., Locke, J. Y., Ma, L.-C., Maglaqui, M., Mao, L., Mitra, S., Patel, D., Rossi, P., Sahdev, S., Sharma, S., Shastry, R., Swapna, G. V. T., Tong, S. N., Wang, D., Wang, H., Zhao, L., Montelione, G. T., and Acton, T. B. (2010) The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium J. Struct. Biol. 172, 21– 33
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    Luft, J. R., Wolfley, J. R., Said, M. I., Nagel, R. M., Lauricella, A. M., Smith, J. L., Thayer, M. H., Veatch, C. K., Snell, E. H., Malkowski, M. G., and Detitta, G. T. (2007) Efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature Protein Sci. Publ. Protein Soc. 16, 715– 722
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    Efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature
    Luft, Joseph R.; Wolfley, Jennifer R.; Said, Meriem I.; Nagel, Raymond M.; Lauricella, Angela M.; Smith, Jennifer L.; Thayer, Max H.; Veatch, Christina K.; Snell, Edward H.; Malkowski, Michael G.; Detitta, George T.
    Protein Science (2007), 16 (4), 715-722CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
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  • Abstract

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    Figure 1

    Figure 1. (A) Selected structures of naturally occurring 10-membered enediynes. The “warhead” is highlighted in red. (B) Proposed mechanism of cycloaromatization of 1 and its effect on DNA scission and CalC self-sacrifice mechanism.

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    Figure 2

    Figure 2. Structure of CalU16. (A) Overlay of NMR (green) and monomers of the crystal (brick- red) structures of CalU16. The N- and C-termini of the protein are labeled, while the dynamic loop is colored yellow. (B) Monomer with secondary structural elements labeled. The residues in the hydrophobic cavity are represented as stick models. (C) B-factors for the Cα atoms in the crystal structure (left) and Cα RMSD values from the NMR ensemble (right) are mapped to the color and tube diameter of “putty” traces showing the general agreement (correlation coefficient 0.559) between the structures.

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    Figure 3

    Figure 3. CalU16 structural homologues. (A) CalU16 (PDB: 4FPW); (B) CalC (PDB: 2L65), calicheamicin resistance protein; (C) TcmN Aro/Cyc (in complex with trans-dihydroquercetin; PDB: 3TVQ) involved in the biosynthesis of tetracenomycin; (D) Hyp-1 (in complex with ethylene glycol; PDB: 3IE5) involved in the biosynthesis of hypericin; (E) NCS (in complex with hydroxybenzaldehyde; PDB: 2VQ5) involved in the biosynthesis of norcoclaurine.

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    Figure 4

    Figure 4. CalU16 and CalU19 assays. Serial disc dilutions of 1 against (A) pSE28a-E. coli (control), (B) pSECalU16-E. coli (CalU16), (C) pSECalU19-E. coli (CalU19), (D) pJB2011-E. coli (CalC). Amount of 1 on discs 1–6 are 10 μg, 1 μg, 100 ng, 50 ng, 10 ng, and 1 ng, respectively. Coomassie-stained 8–12% SDS-PAGE gradient gel of (E) CalU16 and (F) CalU19 in the presence of DTT (lane 1), 1 (lane 2), and DTT and 1 (lane 3).

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    Figure 5

    Figure 5. Site directed mutagenesis of CalU16 and CalU19. Docking models of (A) CalC (B) CalU16, and (C) CalU19 with mutated glycine residues represented as spheres where colored Gly residues indicate cleavage sites, wheat Gly residues indicate mutations that did not affect activity and calicheamicin (1) is represented as a stick model. Results of disc diffusion assay in CalU16 mutants (D) pSE28a-E. coli (control); (E) pSEU16G128V-E. coli; (F) pSEU16G128R-E. coli; (G) pSECalU16-E. coli; and CalU19 mutants (H) pSE28a-E. coli (control); (I) pSEU19G177V-E. coli; (J) pSEU19G177R-E. coli; (K) pSECalU19-E. coli. Amount of 1 on discs 1–6 are 10 μg, 1 μg, 100 ng, 50 ng, 10 ng, and 1 ng, respectively.

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  • References

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      Understanding and exploiting nature's chemical arsenal: the past, present and future of calicheamicin research
      Thorson, Jon S.; Sievers, Eric L.; Ahlert, Joachim; Shepard, Erica; Whitwam, Ross E.; Onwueme, Kenolisa C.; Ruppen, Mark
      Current Pharmaceutical Design (2000), 6 (18), 1841-1879CODEN: CPDEFP; ISSN:1381-6128. (Bentham Science Publishers)
      A review with 234 refs. The enediyne antitumor antibiotics are appreciated for their novel mol. architecture, their remarkable biol. activity and their fascinating mode of action and many have spawned considerable interest as anticancer agents in the pharmaceutical industry. Of equal importance to these astonishing properties, the enediynes also offer a distinct opportunity to study the unparalleled biosyntheses of their unique mol. scaffolds and what promises to be unprecedented modes of self-resistance to highly reactive natural products. Elucidation of these aspects should unveil novel mechanistic enzymol., and may provide access to the rational biosynthetic modification of enediyne structure for new drug leads, the construction of enediyne overproducing strains and eventually lead to an enediyne combinatorial biosynthesis program. This article strives to compile and present the crit. research discoveries relevant to the clin. most promising enediyne, calicheamicin, from a historical perspective. Recent progress, particularly in the areas of biosynthesis, self-resistance, bio-engineering analogs and clin. studies are also highlighted.
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      Galm, U., Hager, M. H., Van Lanen, S. G., Ju, J., Thorson, J. S., and Shen, B. (2005) Antitumor antibiotics: Bleomycin, enediynes, and mitomycin Chem. Rev. 105, 739– 758
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      Antitumor Antibiotics: Bleomycin, Enediynes, and Mitomycin
      Galm, Ute; Hager, Martin H.; Van Lanen, Steven G.; Ju, Jianhua; Thorson, Jon S.; Shen, Ben
      Chemical Reviews (Washington, DC, United States) (2005), 105 (2), 739-758CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The review discusses discovery, biol. activities and resistance to bleomycin, enediynes, and mitomycin.
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      Liang, Z.-X. (2010) Complexity and simplicity in the biosynthesis of enediyne natural products Nat. Prod. Rep. 27, 499– 528
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      Complexity and simplicity in the biosynthesis of enediyne natural products
      Liang, Zhao-Xun
      Natural Product Reports (2010), 27 (4), 499-528CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)
      A review. The review highlights the recent progress on the elucidation of the biosynthetic mechanism of enediyne natural products. A large body of knowledge has been gathered from the genetic, biochem. and structural studies since the sequencing of the first two enediyne gene clusters in 2002. Primary attention is devoted to the novel pathways and enzymes in the biosynthesis of the enediyne warhead as well as the peripheral moieties.
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      Characterization of the in vitro cyclization chemistry of calicheamicin and its relation to DNA cleavage
      De Voss, James J.; Hangeland, Jon J.; Townsend, Craig A.
      Journal of the American Chemical Society (1990), 112 (11), 4554-6CODEN: JACSAT; ISSN:0002-7863.
      The proposed mechanism of thiol activation of calicheamicin γ1I (I), one of the diynene antitumor antibiotics, was examd. in variable-temp. NMR studies. Sep. reactions of the drug with Me thioglycolate and tri-n-butylphosphine led to the identification and characterization of a reactive intermediate resulting from cleavage of the allylic methyltrisulfide and intramol. β-addn. of the resulting thio(ate) to the enone system. This intermediate undergoes 1st-order decompn. at a convenient rate at about -10°. The end product of this process contains 2 H atoms abstracted from the medium consistent with the intervention of a 1,4-diyl formed in the proposed Bergman rearrangement. The relatively long soln. half-life of this intermediate (calcd. to be 4.5 s at 37° in conjunction with other findings led to the formulation of a kinetic scheme to evaluate thermodn. and kinetic factors in the sequence-selective cleavages of DNA characteristic of calicheamicin.
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      Chatterjee, M., Cramer, K. D., and Townsend, C. A. (1993) Kinetic nature of thiol activation in DNA cleavage by calicheamicin J. Am. Chem. Soc. 115, 3374– 3375
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      Myers, A. G., Cohen, S. B., and Kwon, B. M. (1994) A study of the reaction of calicheamicin γ 1 with glutathione in the presence of double-stranded DNA J. Am. Chem. Soc. 116, 1255– 1271
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      Zein, N., Sinha, A. M., McGahren, W. J., and Ellestad, G. A. (1988) Calicheamicin γ 1I: An antitumor antibiotic that cleaves double-stranded DNA site specifically Science 240, 1198– 1201
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      Zein, N., Poncin, M., Nilakantan, R., and Ellestad, G. A. (1989) Calicheamicin γ 1I and DNA: Molecular recognition process responsible for site-specificity Science 244, 697– 699
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      Mah, S. C., Price, M. A., Townsend, C. A., and Tullius, T. D. (1994) Features of DNA recognition for oriented binding and cleavage by calicheamicin Tetrahedron 50, 1361– 1378
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      Biggins, J. B., Prudent, J. R., Marshall, D. J., and Thorson, J. S. (2006) A continuous assay for DNA cleavage using molecular break lights Methods Mol. Biol. 335, 83– 92
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      A continuous assay for DNA cleavage using molecular break lights
      Biggins, John B.; Prudent, James R.; Marshall, David J.; Thorson, Jon S.
      Methods in Molecular Biology (Totowa, NJ, United States) (2006), 335 (Fluorescent Energy Transfer Nucleic Acid Probes), 83-92CODEN: MMBIED; ISSN:1064-3745. (Humana Press Inc.)
      Exploring the properties of mols. that cleave DNA (i.e., enzymic nucleases, chem. footprinting agents, and naturally occurring DNA cleaving antibiotics) has been an ongoing process with benefits extending toward both lab. and clin. applications. Despite the progress that has been made toward understanding the mechanics of DNA cleavage, a simple and continuous assay for detecting DNA cleavage has been lacking. Herein, the authors describe the mol. break light assay, wherein a single oligonucleotide modified by a 5'-fluorophore-3'-quencher pair adopting a stem-loop structure with an appropriate DNA recognition site, provides for the rapid assaying of DNA cleavage with high sensitivity. Furthermore, the described methodol. is highly convenient in that it is readily adaptable to common lab. fluorometers and multi-well plate/array systems, which may provide the basis for high-throughput screening of novel DNA cleaving agents. This assay may also be further extended to natural or "unnatural" transcription factor protection assay systems.
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      Danishefsky, S. J. and Shair, M. D. (1996) Observations in the chemistry and biology of cyclic enediyne antibiotics: Total syntheses of calicheamicin γ1I and dynemicin A J. Org. Chem. 61, 16– 44
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      Nicolaou, K. C., Hale, C. R. H., and Nilewski, C. (2012) A total synthesis trilogy: Calicheamicin γ1(I), Taxol, and brevetoxin A Chem. Rec. 12, 407– 441
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      A total synthesis trilogy: Calicheamicin γ1I, Taxol and brevetoxin A
      Nicolaou, K. C.; Hale, Christopher R. H.; Nilewski, Christian
      Chemical Record (2012), 12 (4), 407-441CODEN: CRHEAK; ISSN:1527-8999. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Detailed behind-the-scenes accounts of the total syntheses of calicheamicin γ1I, Taxol and brevetoxin A are discussed with particular emphasis placed on strategies and tactics employed in these campaigns. DOI 10.1002/tcr.201200005.
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      Ahlert, J., Shepard, E., Lomovskaya, N., Zazopoulos, E., Staffa, A., Bachmann, B. O., Huang, K., Fonstein, L., Czisny, A., Whitwam, R. E., Farnet, C. M., and Thorson, J. S. (2002) The calicheamicin gene cluster and its iterative type I enediyne PKS Science 297, 1173– 1176
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      The calicheamicin gene cluster and its iterative type I enediyne PKS
      Ahlert, Joachim; Shepard, Erica; Lomovskaya, Natalia; Zazopoulos, Emmanuel; Staffa, Alfredo; Bachmann, Brian O.; Huang, Kexue; Fonstein, Leonid; Czisny, Anne; Whitwam, Ross E.; Farnet, Chris M.; Thorson, Jon S.
      Science (Washington, DC, United States) (2002), 297 (5584), 1173-1176CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The enediynes exemplify nature's ingenuity. We have cloned and characterized the biosynthetic locus coding for perhaps the most notorious member of the nonchromoprotein enediyne family, calicheamicin. This gene cluster contains an unusual polyketide synthase (PKS) that is demonstrated to be essential for enediyne biosynthesis. Comparison of the calicheamicin locus with the locus encoding the chromoprotein enediyne C-1027 reveals that the enediyne PKS is highly conserved among these distinct enediyne families. Contrary to previous hypotheses, this suggests that the chromoprotein and nonchromoprotein enediynes are generated by similar biosynthetic pathways.
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      Liu, W., Ahlert, J., Gao, Q., Wendt-Pienkowski, E., Shen, B., and Thorson, J. S. (2003) Rapid PCR amplification of minimal enediyne polyketide synthase cassettes leads to a predictive familial classification model Proc. Natl. Acad. Sci. U.S.A. 100, 11959– 11963
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      Rapid PCR amplification of minimal enediyne polyketide synthase cassettes leads to a predictive familial classification model
      Liu, Wen; Ahlert, Joachim; Gao, Qunjie; Wendt-Pienkowski, Evelyn; Shen, Ben; Thorson, Jon S.
      Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (21), 11959-11963CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      A universal PCR method for the rapid amplification of minimal enediyne polyketide synthase (PKS) genes and the application of this methodol. to clone remaining prototypical genes from producers of structurally detd. enediynes in both family types are presented. A phylogenetic anal. of the new pool of bona fide enediyne PKS genes, consisting of three from 9-membered producers (neocarzinostatin, C1027, and maduropeptin) and three from 10-membered producers (calicheamicin, dynemicin, and esperamicin), reveals a clear genotypic distinction between the two structural families from which to form a predictive model. The results from this study support the postulation that the minimal enediyne PKS helps define the structural divergence of the enediyne core and provides the key tools for generating enediyne hybrid genes/mol. scaffolds; by using the model, a classification is also provided for the unknown enediyne PKS genes previously identified via genome scanning.
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      Biggins, J. B., Onwueme, K. C., and Thorson, J. S. (2003) Resistance to enediyne antitumor antibiotics by CalC self-sacrifice Science 301, 1537– 1541
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      Singh, S., Hager, M. H., Zhang, C., Griffith, B. R., Lee, M. S., Hallenga, K., Markley, J. L., and Thorson, J. S. (2006) Structural insight into the self-sacrifice mechanism of enediyne resistance ACS Chem. Biol. 1, 451– 460
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      Structural insight into the self-sacrifice mechanism of enediyne resistance
      Singh, Shanteri; Hager, Martin H.; Zhang, Changsheng; Griffith, Byron R.; Lee, Min S.; Hallenga, Klaas; Markley, John L.; Thorson, Jon S.
      ACS Chemical Biology (2006), 1 (7), 451-460CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      The recent discovery of the first "self-sacrifice" mechanism for bacterial resistance to the enediyne antitumor antibiotics, where enediyne-induced proteolysis of the resistance protein CalC inactivates both the highly reactive metabolite and the resistance protein, revealed yet another ingenious bacterial mechanism for controlling reactive metabolites. As reported herein, the first 3D structures of CalC and CalC in complex with calicheamicin (CLM) divulge CalC to be a member of the steroidogenic acute regulatory protein (StAR)-related transfer (START) domain superfamily. In contrast to previous studies of proteins known to bind DNA-damaging natural products (e.g., bleomycins, mitomycins, and 9-membered chromoprotein enediynes), this is the 1st demonstrated involvement of a START domain fold. Consistent with the CalC self-sacrifice mechanism, CLM in complex with CalC is positioned for direct H abstraction from Gly113 to initiate the oxidative proteolysis-based resistance mechanism. These structural studies also illuminate, for the 1st time, a small DNA-binding region within CalC that may serve to localize CalC to the enediyne target (DNA). Given the role of START domains in nuclear/cytosolic transport and translocation, this structural study also may implicate START domains as post-endocytotic intracellular chaperones for enediyne-based therapeutics such as MyloTarg.
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    20. 20
      Zhang, C., Griffith, B. R., Fu, Q., Albermann, C., Fu, X., Lee, I.-K., Li, L., and Thorson, J. S. (2006) Exploiting the reversibility of natural product glycosyltransferase-catalyzed reactions Science 313, 1291– 1294
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      20
      Exploiting the Reversibility of Natural Product Glycosyltransferase-Catalyzed Reactions
      Zhang, Changsheng; Griffith, Byron R.; Fu, Qiang; Albermann, Christoph; Fu, Xun; Lee, In-Kyoung; Li, Lingjun; Thorson, Jon S.
      Science (Washington, DC, United States) (2006), 313 (5791), 1291-1294CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Glycosyltransferases (GTs), an essential class of ubiquitous enzymes, are generally perceived as unidirectional catalysts. In contrast, we report that four glycosyltransferases from two distinct natural product biosynthetic pathways-calicheamicin and vancomycin-readily catalyze reversible reactions, allowing sugars and aglycons to be exchanged with ease. As proof of the broader applicability of these new reactions, more than 70 differentially glycosylated calicheamicin and vancomycin variants are reported. This study suggests the reversibility of GT-catalyzed reactions may be general and useful for generating exotic nucleotide sugars, establishing in vitro GT activity in complex systems, and enhancing natural product diversity.
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      Johnson, H. D. and Thorson, J. S. (2008) Characterization of CalE10, the N-oxidase involved in calicheamicin hydroxyaminosugar formation J. Am. Chem. Soc. 130, 17662– 17663
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      Belecki, K., Crawford, J. M., and Townsend, C. A. (2009) Production of octaketide polyenes by the calicheamicin polyketide synthase CalE8: Implications for the biosynthesis of enediyne core structures J. Am. Chem. Soc. 131, 12564– 12566
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      Production of Octaketide Polyenes by the Calicheamicin Polyketide Synthase CalE8: Implications for the Biosynthesis of Enediyne Core Structures
      Belecki, Katherine; Crawford, Jason M.; Townsend, Craig A.
      Journal of the American Chemical Society (2009), 131 (35), 12564-12566CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Enediyne antibiotics are categorized according to the presence of either a 9- or 10-membered ring within their polyketide-derived core structures. Recent literature reports have favored the notion that biosynthetic divergence of the two structural families is detd. by the enediyne polyketide synthases (PKSs) alone. We now disclose the simultaneous in vitro prodn. of three octaketide polyenes by biosynthetic enzymes for the 10-membered enediyne calicheamicin γ1I, including the elusive β-keto acid precursor to a previously described C15 Me hexaenone. Alongside these two polyene products, we have addnl. detected a hydrocarbon heptaene previously isolated only from 9-membered enediyne systems. The discovery of the heptaene in the calicheamicin system promotes a more convergent model for the early steps of enediyne biosynthesis. Furthermore, the synthesis of this set of octaketides by the enediyne PKS CalE8 and thioesterase CalE7 suggests, in contrast to recent biosynthetic proposals, that accessory enzymes may be necessary to initiate differentiation to 9- or 10-membered enediyne precursors, either by modulation of enediyne PKS activity or by interception and modification of polyketide chain-extension intermediates.
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      Horsman, G. P., Chen, Y., Thorson, J. S., and Shen, B. (2010) Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes Proc. Natl. Acad. Sci. U.S.A. 107, 11331– 11335
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      Gantt, R. W., Peltier-Pain, P., Singh, S., Zhou, M., and Thorson, J. S. (2013) Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis Proc. Natl. Acad. Sci. U.S.A. 110, 7648– 7653
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      Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis
      Gantt, Richard W.; Peltier-Pain, Pauline; Singh, Shanteri; Zhou, Maoquan; Thorson, Jon S.
      Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (19), 7648-7653, S7648/1-S7648/72CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed max. improvements in kcat/Km of >400-fold and >15-fold for formation of NDP-glucoses and UDP-sugars, resp. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP-sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.
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      Belecki, K. and Townsend, C. A. (2012) Environmental control of the calicheamicin polyketide synthase leads to detection of a programmed octaketide and a proposal for enediyne biosynthesis Angew. Chem., Int. Ed. 51, 11316– 11319
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      Belecki, K. and Townsend, C. A. (2013) Biochemical determination of enzyme-bound metabolites: Preferential accumulation of a programmed octaketide on the enediyne polyketide synthase CalE8 J. Am. Chem. Soc. 135, 14339– 14348
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      Ricart, A. D. (2011) Antibody-drug conjugates of calicheamicin derivative: Gemtuzumab Ozogamicin and Inotuzumab Ozogamicin Clin. Cancer Res. 17, 6417– 6427
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      Antibody-Drug Conjugates of Calicheamicin Derivative: Gemtuzumab Ozogamicin and Inotuzumab Ozogamicin
      Ricart, Alejandro D.
      Clinical Cancer Research (2011), 17 (20), 6417-6427CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
      A review. Antibody-drug conjugates (ADC) are an attractive approach for the treatment of acute myeloid leukemia and non-Hodgkin lymphomas, which in most cases, are inherently sensitive to cytotoxic agents. CD33 and CD22 are specific markers of myeloid leukemias and B-cell malignancies, resp. These endocytic receptors are ideal for an ADC strategy because they can effectively carry the cytotoxic payload into the cell. Gemtuzumab ozogamicin (GO, Mylotarg) and inotuzumab ozogamicin consist of a deriv. of calicheamicin (a potent DNA-binding cytotoxic antibiotic) linked to a humanized monoclonal IgG4 antibody directed against CD33 or CD22, resp. Both of these ADCs have a target-mediated pharmacokinetic disposition. GO was the first drug to prove the ADC concept in the clinic, specifically in phase II studies that included substantial proportions of older patients with relapsed acute myeloid leukemia. In contrast, in phase III studies, it has thus far failed to show clin. benefit in first-line treatment in combination with std. chemotherapy. Inotuzumab ozogamicin has shown remarkable clin. activity in relapsed/refractory B-cell non-Hodgkin lymphoma, and it has started phase III evaluation. The safety profile of these ADCs includes reversible myelosuppression (esp. neutropenia and thrombocytopenia), elevated hepatic transaminases, and hyperbilirubinemia. There have been postmarketing reports of hepatotoxicity, esp. veno-occlusive disease, assocd. with GO. The incidence is ∼2%, but patients who undergo hematopoietic stem cell transplantation have an increased risk. As we steadily move toward the goal of personalized medicine, these kinds of agents will provide a unique opportunity to treat selected patient subpopulations based on the expression of their specific tumor targets. Clin Cancer Res; 17(20); 6417-27.
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      Trail, P. A. (2013) Antibody drug conjugates as cancer therapeutics Antibodies 2, 113– 129
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      Krissinel, E. and Henrick, K. (2007) Inference of macromolecular assemblies from crystalline state J. Mol. Biol. 372, 774– 797
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      Inference of Macromolecular Assemblies from Crystalline State
      Krissinel, Evgeny; Henrick, Kim
      Journal of Molecular Biology (2007), 372 (3), 774-797CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      The authors discuss basic phys.-chem. principles underlying the formation of stable macromol. complexes, which in many cases are likely to be the biol. units performing a certain physiol. function. The authors also consider available theor. approaches to the calcn. of macromol. affinity and entropy of complexation. The latter is shown to play an important role and make a major effect on complex size and symmetry. The authors develop a new method, based on chem. thermodn., for automatic detection of macromol. assemblies in the Protein Data Bank (PDB) entries that are the results of x-ray diffraction expts. As found, biol. units may be recovered at 80-90% success rate, which makes x-ray crystallog. an important source of exptl. data on macromol. complexes and protein-protein interactions. The method is implemented as a public WWW service (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html).
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      Iyer, L. M., Koonin, E. V., and Aravind, L. (2001) Adaptations of the helix-grip fold for ligand binding and catalysis in the START domain superfamily Proteins 43, 134– 144
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      Thorsell, A.-G., Lee, W. H., Persson, C., Siponen, M. I., Nilsson, M., Busam, R. D., Kotenyova, T., Schüler, H., and Lehtiö, L. (2011) Comparative structural analysis of lipid binding START domains PloS One 6, e19521
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      Alpy, F. and Tomasetto, C. (2005) Give lipids a START: The StAR-related lipid transfer (START) domain in mammals J. Cell Sci. 118, 2791– 2801
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      Give lipids a START: The StAR-related lipid transfer (START) domain in mammals
      Alpy, Fabien; Tomasetto, Catherine
      Journal of Cell Science (2005), 118 (13), 2791-2801CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)
      A review. The steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain is a protein module of ∼210 residues that binds lipids, including sterols. Fifteen mammalian proteins, STARD1-STARD15, possess a START domain and these can be grouped into 6 subfamilies. Cholesterol, 25-hydroxycholesterol, phosphatidylcholine, phosphatidylethanolamine, and ceramides are ligands for STARD1/STARD3/STARD5, STARD5, STARD2/STARD10, STARD10 and STARD11, resp. The lipids or sterols bound by the remaining 9 START proteins are unknown. Recent studies have shown that the C-terminal end of the domain plays a fundamental role, forming a lid over a deep lipid-binding pocket that shields the ligand from the external environment. The START domain can be regarded as a lipid-exchange and/or a lipid-sensing domain. Mammalian START proteins have diverse expression patterns and can be found free in the cytoplasm, attached to membranes or in the nucleus. They appear to function in a variety of distinct physiol. processes, such as lipid transfer between intracellular compartments, lipid metab., and modulation of signaling events. Mutation or misexpression of START proteins is linked to pathol. processes, including genetic disorders, autoimmune disease, and cancer.
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      Holm, L. and Rosenström, P. (2010) Dali server: Conservation mapping in 3D Nucleic Acids Res. 38, W545– 549
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      Dali server: conservation mapping in 3D
      Holm, Liisa; Rosenstrom, Paivi
      Nucleic Acids Research (2010), 38 (Web Server), W545-W549CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Our web site (http://ekhidna.biocenter.helsinki.fi/daliserver) runs the Dali program for protein structure comparison. The web site consists of three parts: (i) the Dali server compares newly solved structures against structures in the Protein Data Bank (PDB), (ii) the Dali database allows browsing precomputed structural neighborhoods and (iii) the pairwise comparison generates suboptimal alignments for a pair of structures. Each part has its own query form and a common format for the results page. The inputs are either PDB identifiers or novel structures uploaded by the user. The results pages are hyperlinked to aid interactive anal. The web interface is simple and easy to use. The key purpose of interactive anal. is to check whether conserved residues line up in multiple structural alignments and how conserved residues and ligands cluster together in multiple structure superimpositions. In favorable cases, protein structure comparison can lead to evolutionary discoveries not detected by sequence anal.
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      Krissinel, E. and Henrick, K. (2004) Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2256– 2268
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      Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions
      Krissinel, E.; Henrick, K.
      Acta Crystallographica, Section D: Biological Crystallography (2004), D60 (12, Pt. 1), 2256-2268CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
      The present paper describes the SSM algorithm of protein structure comparison in three dimensions, which includes an original procedure of matching graphs built on the protein's secondary-structure elements, followed by an iterative three-dimensional alignment of protein backbone Cα atoms. The SSM results are compared with those obtained from other protein comparison servers, and the advantages and disadvantages of different scores that are used for structure recognition are discussed. A new score, balancing the r.m.s.d. and alignment length Nalign, is proposed. It is found that different servers agree reasonably well on the new score, while showing considerable differences in r.m.s.d. and Nalign.
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      Ames, B. D., Korman, T. P., Zhang, W., Smith, P., Vu, T., Tang, Y., and Tsai, S.-C. (2008) Crystal structure and functional analysis of tetracenomycin ARO/CYC: Implications for cyclization specificity of aromatic polyketides Proc. Natl. Acad. Sci. U.S.A. 105, 5349– 5354
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      Michalska, K., Fernandes, H., Sikorski, M., and Jaskolski, M. (2010) Crystal structure of Hyp-1, a St. John’s Wort protein implicated in the biosynthesis of hypericin J. Struct. Biol. 169, 161– 171
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      Ilari, A., Franceschini, S., Bonamore, A., Arenghi, F., Botta, B., Macone, A., Pasquo, A., Bellucci, L., and Boffi, A. (2009) Structural basis of enzymatic (S)-norcoclaurine biosynthesis J. Biol. Chem. 284, 897– 904
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      Structural basis of enzymatic (S)-norcoclaurine biosynthesis
      Ilari, Andrea; Franceschini, Stefano; Bonamore, Alessandra; Arenghi, Fabio; Botta, Bruno; Macone, Alberto; Pasquo, Alessandra; Bellucci, Luca; Boffi, Alberto
      Journal of Biological Chemistry (2009), 284 (2), 897-904CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Norcoclaurine synthase (NCS) catalyzes the stereospecific Pictet-Spengler cyclization between dopamine and 4-hydroxyphenylacetaldehyde, the key step in the benzylisoquinoline alkaloid biosynthetic pathway. Here, the crystallog. structure of NCS from Thalictrum flavum in its complex with its substrate, dopamine, and the nonreactive substrate analog, 4-hydroxybenzaldehyde, was solved at 2.1 Å resoln. NCS shared no common features with the functionally correlated "Pictet-Spenglerases" that catalyze the 1st step of the indole alkaloid pathways and conformed to the overall fold of the Bet v1-like protein. The active site of NCS was located within a 20-Å-long catalytic tunnel and was shaped by the side-chains of the Tyr-108, Lys-122, Asp-141, and Glu-110 residues. The geometry of the amino acid side-chains with respect to the substrates revealed the structural determinants that govern the mechanism of the stereoselective Pictet-Spengler cyclization, thus establishing an excellent foundation for the understanding of the finer details of the catalytic process. Site-directed mutations of the relevant residues confirmed the assignment based on the crystallog. findings.
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      Kofler, S., Asam, C., Eckhard, U., Wallner, M., Ferreira, F., and Brandstetter, H. (2012) Crystallographically mapped ligand binding differs in high and low IgE binding isoforms of birch pollen allergen bet v 1 J. Mol. Biol. 422, 109– 123
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      Li, W., Wang, L., Sheng, X., Yan, C., Zhou, R., Hang, J., Yin, P., and Yan, N. (2013) Molecular basis for the selective and ABA-independent inhibition of PP2CA by PYL13 Cell Res. 23, 1369– 1379
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      Molecular basis for the selective and ABA-independent inhibition of PP2CA by PYL13
      Li, Wenqi; Wang, Li; Sheng, Xinlei; Yan, Chuangye; Zhou, Rui; Hang, Jing; Yin, Ping; Yan, Nieng
      Cell Research (2013), 23 (12), 1369-1379CODEN: CREEB6; ISSN:1001-0602. (NPG Nature Asia-Pacific)
      PYR1/PYL/RCAR family proteins (PYLs) are well-characterized abscisic acid (ABA) receptors. Among the 14 PYL members in Arabidopsis thaliana, PYL13 is ABA irresponsive and its function has remained elusive. Here, we show that PYL13 selectively inhibits the phosphatase activity of PP2CA independent of ABA. The crystal structure of PYL13-PP2CA complex, which was detd. at 2.4 Å resoln., elucidates the mol. basis for the specific recognition between PP2CA and PYL13. In addn. to the canonical interactions between PYLs and PP2Cs, an extra interface is identified involving an element in the vicinity of a previously uncharacterized CCCH zinc-finger (ZF) motif in PP2CA. Sequence blast identified another 56 ZF-contg. PP2Cs, all of which are from plants. The structure also reveals the mol. determinants for the ABA irresponsiveness of PYL13. Finally, biochem. anal. suggests that PYL13 may hetero-oligomerize with PYL10. These two PYLs antagonize each other in their resp. ABA-independent inhibitions of PP2Cs. The biochem. and structural studies provide important insights into the function of PYL13 in the stress response of plant and set up a foundation for future biotechnol. applications of PYL13.
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      Pasternak, O., Bujacz, G. D., Fujimoto, Y., Hashimoto, Y., Jelen, F., Otlewski, J., Sikorski, M. M., and Jaskolski, M. (2006) Crystal structure of Vigna radiata cytokinin-specific binding protein in complex with zeatin Plant Cell 18, 2622– 2634
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      Schirmer, T., Hoffimann-Sommergrube, K., Susani, M., Breiteneder, H., and Marković-Housley, Z. (2005) Crystal structure of the major celery allergen Api g 1: Molecular analysis of cross-reactivity J. Mol. Biol. 351, 1101– 1109
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      Osipiuk, J., Wu, R., Moy, S., and Joachimiak, A. (2006) X-ray crystal structure of conserved hypothetical protein EF_2215 from . PDB ID: 2NN5. Unpublished.
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      Singarapu, K., Eletsky, A., Sathyamoorthy, B., Sukumaran, D., Wang, D., Jiang, M., Ciccosanti, C., Xiao, R., Liu, J., Baran, M. C., Swapna, G., Acton, T. B., Rost, B., Montelione, G. T., and Szyperski, T. (2008) Solution NMR structure of protein encoded by gene BPP1335 from . PDB ID: 2K5G. Unpublished.
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      Clark, B. J. (2012) The mammalian START domain protein family in lipid transport in health and disease J. Endocrinol. 212, 257– 275
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      The mammalian START domain protein family in lipid transport in health and disease
      Clark, Barbara J.
      Journal of Endocrinology (2012), 212 (3), 257-275CODEN: JOENAK; ISSN:0022-0795. (BioScientifica Ltd.)
      A review. Lipid transfer proteins of the steroidogenic acute regulatory protein-related lipid transfer (START) domain family are defined by the presence of a conserved ∼210 amino acid sequence that folds into an α/β helix-grip structure forming a hydrophobic pocket for ligand binding. The mammalian START proteins bind diverse ligands, such as cholesterol, oxysterols, phospholipids, sphingolipids, and possibly fatty acids, and have putative roles in non-vesicular lipid transport, thioesterase enzymic activity, and tumor suppression. However, the biol. functions of many members of the START domain protein family are not well established. Recent research has focused on characterizing the cell-type distribution and regulation of the START proteins, examg. the specificity and directionality of lipid transport, and identifying disease states assocd. with dysregulation of START protein expression. This review summarizes the current concepts of the proposed physiol. and pathol. roles for the mammalian START domain proteins in cholesterol and lipid trafficking.
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      Chang, A., Singh, S., Helmich, K. E., Goff, R. D., Bingman, C. A., Thorson, J. S., and Phillips, G. N., Jr. (2011) Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity Proc. Natl. Acad. Sci. U.S.A. 108, 17649– 17654
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      Gerlt, J. A. and Babbitt, P. C. (2000) Can sequence determine function? Genome Biol. 1, 1– 10
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      Whisstock, J. C. and Lesk, A. M. (2003) Prediction of protein function from protein sequence and structure Q. Rev. Biophys. 36, 307– 340
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      Prediction of protein function from protein sequence and structure
      Whisstock, James C.; Lesk, Arthur M.
      Quarterly Reviews of Biophysics (2003), 36 (3), 307-340CODEN: QURBAW; ISSN:0033-5835. (Cambridge University Press)
      A review. The sequence of a genome contains the plans of the possible life of an organism, but implementation of genetic information depends on the functions of the proteins and nucleic acids that it encodes. Many individual proteins of known sequence and structure present challenges to the understanding of their function. In particular, a no. of genes responsible for diseases have been identified but their specific functions are unknown. Whole-genome sequencing projects are a major source of proteins of unknown function. Annotation of a genome involves assignment of functions to gene products, in most cases on the basis of amino-acid sequence alone. 3D structure can aid the assignment of function, motivating the challenge of structural genomics projects to make structural information available for novel uncharacterized proteins. Structure-based identification of homologs often succeeds where sequence-alone-based methods fail, because in many cases evolution retains the folding pattern long after sequence similarity becomes undetectable. Nevertheless, prediction of protein function from sequence and structure is a difficult problem, because homologous proteins often have different functions. Many methods of function prediction rely on identifying similarity in sequence and/or structure between a protein of unknown function and one or more well-understood proteins. Alternative methods include inferring conservation patterns in members of a functionally uncharacterized family for which many sequences and structures are known. However, these inferences are tenuous. Such methods provide reasonable guesses at function, but are far from foolproof. It is therefore fortunate that the development of whole-organism approaches and comparative genomics permits other approaches to function prediction when the data are available. These include the use of protein-protein interaction patterns, and correlations between occurrences of related proteins in different organisms, as indicators of functional properties. Even if it is possible to ascribe a particular function to a gene product, the protein may have multiple functions. A fundamental problem is that function is in many cases an ill-defined concept. In this article we review the state of the art in function prediction and describe some of the underlying difficulties and successes.
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      Schnoes, A. M., Brown, S. D., Dodevski, I., and Babbitt, P. C. (2009) Annotation error in public databases: Misannotation of molecular function in enzyme superfamilies PLoS Comput. Biol. 5, e1000605
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      Annotation error in public databases: misannotation of molecular function in enzyme superfamilies
      Schnoes Alexandra M; Brown Shoshana D; Dodevski Igor; Babbitt Patricia C
      PLoS computational biology (2009), 5 (12), e1000605 ISSN:.
      Due to the rapid release of new data from genome sequencing projects, the majority of protein sequences in public databases have not been experimentally characterized; rather, sequences are annotated using computational analysis. The level of misannotation and the types of misannotation in large public databases are currently unknown and have not been analyzed in depth. We have investigated the misannotation levels for molecular function in four public protein sequence databases (UniProtKB/Swiss-Prot, GenBank NR, UniProtKB/TrEMBL, and KEGG) for a model set of 37 enzyme families for which extensive experimental information is available. The manually curated database Swiss-Prot shows the lowest annotation error levels (close to 0% for most families); the two other protein sequence databases (GenBank NR and TrEMBL) and the protein sequences in the KEGG pathways database exhibit similar and surprisingly high levels of misannotation that average 5%-63% across the six superfamilies studied. For 10 of the 37 families examined, the level of misannotation in one or more of these databases is >80%. Examination of the NR database over time shows that misannotation has increased from 1993 to 2005. The types of misannotation that were found fall into several categories, most associated with "overprediction" of molecular function. These results suggest that misannotation in enzyme superfamilies containing multiple families that catalyze different reactions is a larger problem than has been recognized. Strategies are suggested for addressing some of the systematic problems contributing to these high levels of misannotation.
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      Baker, D. and Sali, A. (2001) Protein structure prediction and structural genomics Science 294, 93– 96
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      Protein structure prediction and structural genomics
      Baker, David; Sali, Andrej
      Science (Washington, DC, United States) (2001), 294 (5540), 93-96CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Genome sequencing projects are producing linear amino acid sequences, but full understanding of the biol. role of these proteins will require knowledge of their structure and function. Although exptl. structure detn. methods are providing high-resoln. structure information about a subset of the proteins, computational structure prediction methods will provide valuable information for the large fraction of sequences whose structures will not be detd. exptl. The first class of protein structure prediction methods, including threading and comparative modeling, rely on detectable similarity spanning most of the modeled sequence and at least one known structure. The second class of methods, de novo or ab initio methods, predict the structure from sequence alone, without relying on similarity at the fold level between the modeled sequence and any of the known structures. In this Viewpoint, we begin by describing the essential features of the methods, the accuracy of the models, and their application to the prediction and understanding of protein function, both for single proteins and on the scale of whole genomes. We then discuss the important role that protein structure prediction methods play in the growing worldwide effort in structural genomics.
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      Hermann, J. C., Marti-Arbona, R., Fedorov, A. A., Fedorov, E., Almo, S. C., Shoichet, B. K., and Raushel, F. M. (2007) Structure-based activity prediction for an enzyme of unknown function Nature 448, 775– 779
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      Structure-based activity prediction for an enzyme of unknown function
      Hermann, Johannes C.; Marti-Arbona, Ricardo; Fedorov, Alexander A.; Fedorov, Elena; Almo, Steven C.; Shoichet, Brian K.; Raushel, Frank M.
      Nature (London, United Kingdom) (2007), 448 (7155), 775-779CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      With many genomes sequenced, a pressing challenge in biol. is predicting the function of the proteins that the genes encode. When proteins are unrelated to others of known activity, bioinformatics inference for function becomes problematic. It would thus be useful to interrogate protein structures for function directly. Here, we predict the function of an enzyme of unknown activity, Tm0936 from Thermotoga maritima, by docking high-energy intermediate forms of thousands of candidate metabolites. The docking hit list was dominated by adenine analogs, which appeared to undergo C6-deamination. Four of these, including 5-methylthioadenosine and S-adenosylhomocysteine (SAH), were tested as substrates, and three had substantial catalytic rate consts. (105 M-1 s-1). The x-ray crystal structure of the complex between Tm0936 and the product resulting from the deamination of SAH, S-inosylhomocysteine, was detd., and it corresponded closely to the predicted structure. The deaminated products can be further metabolized by T. maritima in a previously uncharacterized SAH degrdn. pathway. Structure-based docking with high-energy forms of potential substrates may be a useful tool to annotate enzymes for function.
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      Guichou, J.-F. and Labesse, G. (2012) Fragment and conquer: From structure to complexes to function Structure 20, 1617– 1619
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      Wright, G. D. (2007) The antibiotic resistome: The nexus of chemical and genetic diversity Nat. Rev. Microbiol. 5, 175– 186
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      The antibiotic resistome: the nexus of chemical and genetic diversity
      Wright, Gerard D.
      Nature Reviews Microbiology (2007), 5 (3), 175-186CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
      A review. Over the millennia, microorganisms have evolved evasion strategies to overcome a myriad of chem. and environmental challenges, including antimicrobial drugs. Even before the first clin. use of antibiotics more than 60 years ago, resistant organisms had been isolated. Moreover, the potential problem of the widespread distribution of antibiotic resistant bacteria was recognized by scientists and health-care specialists from the initial use of these drugs. Why is resistance inevitable and where does it come from. Understanding the mol. diversity that underlies resistance will inform our use of these drugs and guide efforts to develop new efficacious antibiotics.
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      Cundliffe, E. and Demain, A. L. (2010) Avoidance of suicide in antibiotic-producing microbes J. Ind. Microbiol. Biotechnol. 37, 643– 672
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      Avoidance of suicide in antibiotic-producing microbes
      Cundliffe, Eric; Demain, Arnold L.
      Journal of Industrial Microbiology & Biotechnology (2010), 37 (7), 643-672CODEN: JIMBFL; ISSN:1367-5435. (Springer)
      A review. Many microbes synthesize potentially autotoxic antibiotics, mainly as secondary metabolites, against which they need to protect themselves. This is done in various ways, ranging from target-based strategies (i.e., modification of normal drug receptors or de novo synthesis of the latter in drug-resistant form) to the adoption of metabolic shielding and/or efflux strategies that prevent drug-target interactions. These self-defense mechanisms have been studied most intensively in antibiotic-producing prokaryotes, of which the most prolific are the actinomycetes. Only a few documented examples pertain to lower eukaryotes while higher organisms have hardly been addressed in this context. Thus, many plant alkaloids, variously described as herbivore repellents or nitrogen excretion devices, are truly antibiotics-even if toxic to humans. As just one example, bulbs of Narcissus spp. (including the King Alfred daffodil) accumulate narciclasine that binds to the larger subunit of the eukaryotic ribosome and inhibits peptide bond formation. However, ribosomes in the Amaryllidaceae have not been tested for possible resistance to narciclasine and other alkaloids. Clearly, the prevalence of suicide avoidance is likely to extend well beyond the remit of the present article.
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      Allan, C. M., Hill, S., Morvaridi, S., Saiki, R., Johnson, J. S., Liau, W.-S., Hirano, K., Kawashima, T., Ji, Z., Loo, J. A., Shepherd, J. N., and Clarke, C. F. (2013) A conserved START domain coenzyme Q-binding polypeptide is required for efficient Q biosynthesis, respiratory electron transport, and antioxidant function in Saccharomyces cerevisiae Biochim. Biophys. Acta 1831, 776– 791
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      Baker, J. R., Woolfson, D. N., Muskett, F. W., Stoneman, R. G., Urbaniak, M. D., and Caddick, S. (2007) Protein–small molecule interactions in neocarzinostatin, the prototypical enediyne chromoprotein antibiotic ChemBioChem 8, 704– 717
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      Weigel, L. M., Clewell, D. B., Gill, S. R., Clark, N. C., McDougal, L. K., Flannagan, S. E., Kolonay, J. F., Shetty, J., Killgore, G. E., and Tenover, F. C. (2003) Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus Science 302, 1569– 1571
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      Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus
      Weigel, Linda M.; Clewell, Don B.; Gill, Steven R.; Clark, Nancye C.; McDougal, Linda K.; Flannagan, Susan E.; Kolonay, James F.; Shetty, Jyoti; Killgore, George E.; Tenover, Fred C.
      Science (Washington, DC, United States) (2003), 302 (5650), 1569-1571CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Vancomycin is usually reserved for treatment of serious infections, including those caused by multidrug-resistant Staphylococcus aureus. A clin. isolate of S. aureus with high-level resistance to vancomycin (minimal inhibitory concn. = 1024 μg/mL) was isolated in June 2002. This isolate harbored a 57.9-kilobase multiresistance conjugative plasmid within which Tn1546 (vanA) was integrated. Addnl. elements on the plasmid encoded resistance to trimethoprim (dfrA), β-lactams (blaZ), aminoglycosides (aacA-aphD), and disinfectants (qacC). Genetic analyses suggest that the long-anticipated transfer of vancomycin resistance to a methicillin-resistant S. aureus occurred in vivo by interspecies transfer of Tn1546 from a co-isolate of Enterococcus faecalis.
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      Qureshi, N. K., Yin, S., and Boyle-Vavra, S. (2014) The role of the Staphylococcal VraTSR regulatory system on vancomycin resistance and vanA operon expression in vancomycin-resistant Staphylococcus aureus PloS One 9, e85873
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      Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P., and Drummond, A. (2012) Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data Bioinformatics 28, 1647– 1649
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      Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data
      Kearse Matthew; Moir Richard; Wilson Amy; Stones-Havas Steven; Cheung Matthew; Sturrock Shane; Buxton Simon; Cooper Alex; Markowitz Sidney; Duran Chris; Thierer Tobias; Ashton Bruce; Meintjes Peter; Drummond Alexei
      Bioinformatics (Oxford, England) (2012), 28 (12), 1647-9 ISSN:.
      UNLABELLED: The two main functions of bioinformatics are the organization and analysis of biological data using computational resources. Geneious Basic has been designed to be an easy-to-use and flexible desktop software application framework for the organization and analysis of biological data, with a focus on molecular sequences and related data types. It integrates numerous industry-standard discovery analysis tools, with interactive visualizations to generate publication-ready images. One key contribution to researchers in the life sciences is the Geneious public application programming interface (API) that affords the ability to leverage the existing framework of the Geneious Basic software platform for virtually unlimited extension and customization. The result is an increase in the speed and quality of development of computation tools for the life sciences, due to the functionality and graphical user interface available to the developer through the public API. Geneious Basic represents an ideal platform for the bioinformatics community to leverage existing components and to integrate their own specific requirements for the discovery, analysis and visualization of biological data. AVAILABILITY AND IMPLEMENTATION: Binaries and public API freely available for download at http://www.geneious.com/basic, implemented in Java and supported on Linux, Apple OSX and MS Windows. The software is also available from the Bio-Linux package repository at http://nebc.nerc.ac.uk/news/geneiousonbl.
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    60. 60
      Acton, T. B., Xiao, R., Anderson, S., Aramini, J., Buchwald, W. A., Ciccosanti, C., Conover, K., Everett, J., Hamilton, K., Huang, Y. J., Janjua, H., Kornhaber, G., Lau, J., Lee, D. Y., Liu, G., Maglaqui, M., Ma, L., Mao, L., Patel, D., Rossi, P., Sahdev, S., Shastry, R., Swapna, G. V. T., Tang, Y., Tong, S., Wang, D., Wang, H., Zhao, L., and Montelione, G. T. (2011) Preparation of protein samples for NMR structure, function, and small-molecule screening studies Methods Enzymol. 493, 21– 60
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      60
      Preparation of protein samples for NMR structure, function, and small-molecule screening studies
      Acton, Thomas B.; Xiao, Rong; Anderson, Stephen; Aramini, James; Buchwald, William A.; Ciccosanti, Colleen; Conover, Ken; Everett, John; Hamilton, Keith; Huang, Yuanpeng Janet; Janjua, Haleema; Kornhaber, Gregory; Lau, Jessica; Lee, Dong Yup; Liu, Gaohua; Maglaqui, Melissa; Ma, Lichung; Mao, Lei; Patel, Dayaban; Rossi, Paolo; Sahdev, Seema; Shastry, Ritu; Swapna, G. V. T.; Tang, Yeufeng; Tong, Saichiu; Wang, Dongyan; Wang, Huang; Zhao, Li; Montelione, Gaetano T.
      Methods in Enzymology (2011), 493 (Fragment-Based Drug Design), 21-60CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)
      A review. In this chapter, we conc. on the prodn. of high-quality protein samples for NMR studies, in particular, we provide an in-depth description of recent advances in the prodn. of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein prodn. platform of the Northeast Structural Genomics Consortium and outline our high-throughput strategies for producing high-quality protein samples for NMR studies. Our strategy is based on the cloning, expression, and purifn. of 6 × -His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and anal. expression systems, parallel preparative scale fermn., and high-throughput purifn. protocols. The 6 × -His affinity tag allows for a similar two-step purifn. procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (>97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5000 of these proteins have been purified to homogeneity in tens of milligram quantities, resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this chapter describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purifn., and biophys. screening technologies are suitable for implementation in a large individual lab. or by a small group of collaborating investigators for structural biol., functional proteomics, ligand screening, and structural genomics research.
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    61. 61
      Xiao, R., Anderson, S., Aramini, J., Belote, R., Buchwald, W. A., Ciccosanti, C., Conover, K., Everett, J. K., Hamilton, K., Huang, Y. J., Janjua, H., Jiang, M., Kornhaber, G. J., Lee, D. Y., Locke, J. Y., Ma, L.-C., Maglaqui, M., Mao, L., Mitra, S., Patel, D., Rossi, P., Sahdev, S., Sharma, S., Shastry, R., Swapna, G. V. T., Tong, S. N., Wang, D., Wang, H., Zhao, L., Montelione, G. T., and Acton, T. B. (2010) The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium J. Struct. Biol. 172, 21– 33
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      Luft, J. R., Wolfley, J. R., Said, M. I., Nagel, R. M., Lauricella, A. M., Smith, J. L., Thayer, M. H., Veatch, C. K., Snell, E. H., Malkowski, M. G., and Detitta, G. T. (2007) Efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature Protein Sci. Publ. Protein Soc. 16, 715– 722
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      Efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature
      Luft, Joseph R.; Wolfley, Jennifer R.; Said, Meriem I.; Nagel, Raymond M.; Lauricella, Angela M.; Smith, Jennifer L.; Thayer, Max H.; Veatch, Christina K.; Snell, Edward H.; Malkowski, Michael G.; Detitta, George T.
      Protein Science (2007), 16 (4), 715-722CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
      An efficient optimization method for the crystn. of biol. macromols. has been developed and tested. This builds on a successful high-throughput technique for the detn. of initial crystn. conditions. The optimization method takes an initial condition identified through screening and then varies the concn. of the macromol., precipitant, and the growth temp. in a systematic manner. The amt. of sample and no. of steps is minimized and no biochem. reformulation is required. In the current application a robotic liq. handling system enables high-throughput use, but the technique can easily be adapted in a nonautomated setting. This method has been applied successfully for the rapid optimization of crystn. conditions in nine representative cases.
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      Otwinowski, Z. and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode Methods Enzymol. 276, 307– 326
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      63
      Processing of x-ray diffraction data collected in oscillation mode
      Otwinowski, Zbyszek; Minor, Wladek
      Methods in Enzymology (1997), 276 (Macromolecular Crystallography, Part A), 307-326CODEN: MENZAU; ISSN:0076-6879. (Academic)
      Macromol. crystallog. is an iterative process. Rarely do the first crystals provide all the necessary data to solve the biol. problem being studied. Each step benefits from experience learned in previous steps. To monitor the progress, the HKL package provides 2 tools: (1) statistics, both weighted (χ2) and unweighted (R-merge), are provided, and the Bayesian reasoning and multicomponent error model facilitates obtaining the proper error ests. and (2) visualization of the process plays a double role by helping the operator to confirm that the process of data redn., including the resulting statistics, is correct, and allowing one to evaluate problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and redn. has been reached. At that point, the effort should be directed to solving the structure. The methods presented here have been applied to solve a large variety of problems, from inorg. mols. with 5 Å unit cell to rotavirus of 700 Å diam. crystd. in 700 × 1000 × 1400 Å cell. Overall quality of the method was tested by many researchers by successful application of the programs to MAD structure detns.
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    64. 64
      Sheldrick, G. M. (2010) Experimental phasing with SHELXC/D/E: Combining chain tracing with density modification Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 479– 485
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      Experimental phasing with SHELXC/D/E: combining chain tracing with density modification
      Sheldrick, George M.
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 479-485CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      The programs SHELXC, SHELXD and SHELXE are designed to provide simple, robust and efficient exptl. phasing of macromols. by the SAD, MAD, SIR, SIRAS and RIP methods and are particularly suitable for use in automated structure-soln. pipelines. This paper gives a general account of exptl. phasing using these programs and describes the extension of iterative d. modification in SHELXE by the inclusion of automated protein main-chain tracing. This gives a good indication as to whether the structure has been solved and enables interpretable maps to be obtained from poorer starting phases. The autotracing algorithm starts with the location of possible seven-residue α-helixes and common tripeptides. After extension of these fragments in both directions, various criteria are used to decide whether to accept or reject the resulting poly-Ala traces. Noncrystallog. symmetry (NCS) is applied to the traced fragments, not to the d. Further features are the use of a 'no-go' map to prevent the traces from passing through heavy atoms or symmetry elements and a splicing technique to combine the best parts of traces (including those generated by NCS) that partly overlap.
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      Terwilliger, T. C. and Berendzen, J. (1999) Automated MAD and MIR structure solution Acta Crystallogr., Sect. D: Biol. Crystallogr. 55, 849– 861
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      Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 486– 501
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      Features and development of Coot
      Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
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      Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method Acta Crystallogr., Sect. D: Biol. Crystallogr. 53, 240– 255
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      Refinement of macromolecular structures by the maximum-likelihood method
      Murshudov, Garib N.; Vagin, Alexei A.; Dodson, Eleanor J.
      Acta Crystallographica, Section D: Biological Crystallography (1997), D53 (3), 240-255CODEN: ABCRE6; ISSN:0907-4449. (Munksgaard)
      A review with many refs. on the math. basis of max. likelihood. The likelihood function for macromol. structures is extended to include prior phase information and exptl. std. uncertainties. The assumption that different parts of a structure might have different errors is considered. A method for estg. σA using "free" reflections is described and its effects analyzed. The derived equations have been implemented in the program REFMAC. This has been tested on several proteins at different stages of refinement (bacterial α-amylase, cytochrome c', cross-linked insulin and oligopeptide binding protein). The results derived using the max.-likelihood residual are consistently better than those obtained from least-squares refinement.
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    68. 68
      Laskowski, R., MacArthur, M., Moss, D., and Thornton, J. (1993) PROCHECK: A program to check the stereochemical quality of protein structures J. Appl. Crystallogr. 26, 283– 291
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      PROCHECK: a program to check the stereochemical quality of protein structures
      Laskowski, Roman A.; MacArthur, Malcolm W.; Moss, David S.; Thornton, Janet M.
      Journal of Applied Crystallography (1993), 26 (2), 283-91CODEN: JACGAR; ISSN:0021-8898.
      The PROCHECK suite of programs provides a detailed check on the stereochem. of a protein structure. Its outputs comprise a no. of plots in PostScript format and a comprehensive residue-by-residue listing. These give an assessment of the overall quality of the structure as compared with well-refined structures of the same resoln. and also highlight regions that may need further investigation. The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modeled on known structures.
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    69. 69
      Shi, J., Wang, Y., Zeng, L., Wu, Y., Deng, J., Zhang, Q., Lin, Y., Li, J., Kang, T., Tao, M., Rusinova, E., Zhang, G., Wang, C., Zhu, H., Yao, J., Zeng, Y.-X., Evers, B. M., Zhou, M.-M., and Zhou, B. P. (2014) Disrupting the interaction of BRD4 with diacetylated twist suppresses tumorigenesis in basal-like breast cancer Cancer Cell 25, 210– 225
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      Disrupting the Interaction of BRD4 with Diacetylated Twist Suppresses Tumorigenesis in Basal-like Breast Cancer
      Shi, Jian; Wang, Yifan; Zeng, Lei; Wu, Yadi; Deng, Jiong; Zhang, Qiang; Lin, Yiwei; Li, Junlin; Kang, Tiebang; Tao, Min; Rusinova, Elena; Zhang, Guangtao; Wang, Chi; Zhu, Haining; Yao, Jun; Zeng, Yi-Xin; Evers, B. Mark; Zhou, Ming-Ming; Zhou, Binhua P.
      Cancer Cell (2014), 25 (2), 210-225CODEN: CCAECI; ISSN:1535-6108. (Elsevier Inc.)
      Twist is a key transcription activator of epithelial-mesenchymal transition (EMT). It remains unclear how Twist induces gene expression. Here we report a mechanism by which Twist recruits BRD4 to direct WNT5A expression in basal-like breast cancer (BLBC). Twist contains a "histone H4-mimic" GK-X-GK motif that is diacetylated by Tip60. The diacetylated Twist binds the second bromodomain of BRD4, whose first bromodomain interacts with acetylated H4, thereby constructing an activated Twist/BRD4/P-TEFb/RNA-Pol II complex at the WNT5A promoter and enhancer. Pharmacol. inhibition of the Twist-BRD4 assocn. reduced WNT5A expression and suppressed invasion, cancer stem cell (CSC)-like properties, and tumorigenicity of BLBC cells. Our study indicates that the interaction with BRD4 is crit. for the oncogenic function of Twist in BLBC.
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      Zhai, J., Ström, A.-L., Kilty, R., Venkatakrishnan, P., White, J., Everson, W. V., Smart, E. J., and Zhu, H. (2009) Proteomic characterization of lipid raft proteins in amyotrophic lateral sclerosis mouse spinal cord FEBS J. 276, 3308– 3323
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      Chen, V. B., Arendall, W. B., 3rd, Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S., and Richardson, D. C. (2010) MolProbity: All-atom structure validation for macromolecular crystallography Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 12– 21
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      71
      MolProbity: all-atom structure validation for macromolecular crystallography
      Chen, Vincent B.; Arendall, W. Bryan, III; Headd, Jeffrey J.; Keedy, Daniel A.; Immormino, Robert M.; Kapral, Gary J.; Murray, Laura W.; Richardson, Jane S.; Richardson, David C.
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (1), 12-21CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      MolProbity is a structure-validation web service that provides broad-spectrum solidly based evaluation of model quality at both the global and local levels for both proteins and nucleic acids. It relies heavily on the power and sensitivity provided by optimized hydrogen placement and all-atom contact anal., complemented by updated versions of covalent-geometry and torsion-angle criteria. Some of the local corrections can be performed automatically in MolProbity and all of the diagnostics are presented in chart and graphical forms that help guide manual rebuilding. X-ray crystallog. provides a wealth of biol. important mol. data in the form of at. three-dimensional structures of proteins, nucleic acids and increasingly large complexes in multiple forms and states. Advances in automation, in everything from crystn. to data collection to phasing to model building to refinement, have made solving a structure using crystallog. easier than ever. However, despite these improvements, local errors that can affect biol. interpretation are widespread at low resoln. and even high-resoln. structures nearly all contain at least a few local errors such as Ramachandran outliers, flipped branched protein side chains and incorrect sugar puckers. It is crit. both for the crystallographer and for the end user that there are easy and reliable methods to diagnose and correct these sorts of errors in structures. MolProbity is the authors' contribution to helping solve this problem and this article reviews its general capabilities, reports on recent enhancements and usage, and presents evidence that the resulting improvements are now beneficially affecting the global database.
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