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ArticleJanuary 15, 2019

Structure of Prototypic Peptide Transporter DtpA from E. coli in Complex with Valganciclovir Provides Insights into Drug Binding of Human PepT1
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Yonca Ural-Blimke
Yonca Ural-Blimke
Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
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Ali Flayhan
Ali Flayhan
Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
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Jan Strauss
Jan Strauss
Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
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http://orcid.org/0000-0002-6208-791X
Vasileios Rantos
Vasileios Rantos
Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
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Kim Bartels
Kim Bartels
Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
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Rolf Nielsen
Rolf Nielsen
Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
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Els Pardon
Els Pardon
Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels 1050, Belgium
VIB-VUB Center for Structural Biology, VIB, Brussels 1050, Belgium
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Jan Steyaert
Jan Steyaert
Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels 1050, Belgium
VIB-VUB Center for Structural Biology, VIB, Brussels 1050, Belgium
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Jan Kosinski
Jan Kosinski
Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
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Esben M. Quistgaard
Esben M. Quistgaard
Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, SE-17177 Stockholm, Sweden
Department of Molecular Biology and Genetics − DANDRITE, Gustav Wieds Vej 10, Aarhus University, DK-8000 Aarhus C, Denmark
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Christian Löw*
Christian Löw
Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, SE-17177 Stockholm, Sweden
*[email protected]
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http://orcid.org/0000-0003-0764-7483

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Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2019, 141, 6, 2404–2412

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https://doi.org/10.1021/jacs.8b11343

Published January 15, 2019

Abstract

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Members of the solute carrier 15 family (SLC15) transport di- and tripeptides as well as peptidomimetic drugs across the cell membrane. Structures of bacterial homologues have provided valuable information on the binding and transport of their natural substrates, but many do not transport medically relevant drugs. In contrast, a homologue from Escherichia coli, DtpA (dipeptide and tripeptide permease), shows a high similarity to human PepT1 (SLC15A1) in terms of ligand selectivity and transports a similar set of drugs. Here, we present the crystal structure of DtpA in ligand-free form (at 3.30 Å resolution) and in complex with the antiviral prodrug valganciclovir (at 2.65 Å resolution) supported by biochemical data. We show that valganciclovir unexpectedly binds with the ganciclovir moiety mimicking the N-terminal residue of a canonical peptide substrate. On the basis of a homology model we argue that this binding mode also applies to the human PepT1 transporter. Our results provide new insights into the binding mode of prodrugs and will assist the rational design of drugs with improved absorption rates.

Subjects

Introduction

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The proton-dependent oligopeptide transporters (POTs) constitute a subfamily of the major facilitator superfamily (MFS), transporting di/tripeptides and peptide-like compounds coupled to cotransport of protons. Mammalian POTs are also referred to as solute carrier 15 transporters (SLC15s). Human PepT1 (SLC15A1) and PepT2 (SLC15A2) are of great pharmacological interest due to their roles in intestinal uptake and renal reabsorption of not only dietary peptides but also drugs such as β-lactam antibiotics (ceftibuten), angiotensin-converting enzyme inhibitors (enalapril), and antiviral prodrugs (valganciclovir and valacyclovir). (1) The prodrugs valganciclovir and valacyclovir are l-valine derivatives of the guanosine analogs ganciclovir and acyclovir, used for treating cytomegalovirus and herpes simplex virus infections, respectively. Conjugation to valine makes these compounds more soluble and improves their uptake rates in humans by turning them into substrates for PepT1 and PepT2. (2,3) PepT1 and PepT2 have been well characterized in terms of ligand binding, but their structures remain unknown. The bacterial homologue of PepT1, DtpA (dipeptide and tripeptide permease), provides an excellent prototype to understand the molecular mechanism of peptide and drug transport due to the high conservation of the binding site as well as the highly similar substrate specificity profile compared to PepT1. (4,5) However, like for their human homologues, structural insights into relevant drug binding are missing for bacterial peptide transporters. Therefore, we combined biochemical, biophysical, and structural approaches to obtain molecular insights into binding and transport of peptides and drugs of the prototypic DtpA transporter, which provided the basis for structural modeling of the human PepT1 transporter.

Results and Discussion

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To produce well-diffracting crystals of DtpA, we used nanobodies as crystallization chaperones, which can improve crystallization by increasing the thermal stability of the target protein, by locking it in a specific conformation, and by mediating crystal contacts. Four nanobodies were generated, which were confirmed to bind a conformational epitope of DtpA and increased its thermal stability by more than 10 °C (Supplementary Figure 1). Nanobody N00 was essential to obtain well-diffracting crystals. Three structures of the DtpA-N00 complex were determined (Table 1, Supplementary Figure 2), including two 3.30-Å ligand-free DtpA structures from different crystallization conditions (MES or glycine buffer) (Figure 1a) and a 2.65-Å valganciclovir-bound structure (glycine buffer). The higher resolution of the drug-bound structure in comparison to the ligand-free structures may relate to the fact that valganciclovir stabilizes the DtpA-nanobody complex further and reduces its conformational flexibility (Figure 1b).

Figure 1. Structure of the peptide transporter-nanobody complex DtpA-N00, and thermal stability with the prodrugs valganciclovir and valacyclovir. (a) Structure of the DtpA-N00 complex (in MES buffer). N-terminal MFS domain is colored in green, C-terminal MFS domain in orange, HA-HB domain in yellow, and N00 in pink. (b) Thermal stabilization effect of valganciclovir and valacyclovir on DtpA, and chemical structure of the prodrugs. Guanine base is highlighted in pale green and the N-terminal valine residue in light orange. Valganciclovir is a mix of two diastereomers with alternative conformations at the chiral center highlighted with a red star.

Table 1. Crystallographic Data Collection and Refinement Statistics for the Ligand-Free and Valganciclovir-Bound DtpA-N00 Structures^a

	DtpA-N00 in glycine buffer, pdb id 6GS7	DtpA-N00 in MES buffer, pdb id 6GS1	DtpA-N00-Valganciclovir, pdb id 6GS4
data collection
space group	P2₁2₁2₁	P2₁2₁2₁	P2₁2₁2₁
cell dimensions
a, b, c (Å)	55.14, 120.53, 163.43	55.46, 120.72, 163.33	54.94, 120.19, 163.67
α, β, γ (deg)	90, 90, 90	90, 90, 90	90, 90, 90
resolution (Å)	19.94–3.30 (3.42–3.30)	48.16–3.29 (3.41–3.29)	19.68–2.65 (2.74–2.65)
R_merge	21.74 (217.7)	26.73 (186.4)	10.74 (207.7)
R_pim	7.97 (74.81)	11.14 (78.46)	4.65 (86.77)
I/σ	7.04 (0.83)	6.37 (1.06)	10.74 (1.06)
CC 1/2	99.2 (49.4)	99.4 (56.0)	99.8 (46.3)
completeness (%)	99.02 (99.70)	99.34 (99.47)	98.98 (97.29)
redundancy	8.5 (8.8)	6.7 (6.6)	6.4 (6.6)
refinement
resolution (Å)	19.94−3.30 (3.42–3.30)	48.16–3.29 (3.41–3.29)	19.68–2.65 (2.74–2.65)
no. of reflns	16919 (1671)	17219 (1679)	32178 (3122)
R_work/R_free	21.03/24.75	25.11/26.60	21.57/23.96
no. of atoms
protein	4568	4576	4542
ligand/ion			60
water		1	7
clashscore	4.46	6.41	3.57
B-factors
protein	108.69	83.22	89.74
ligand/ion			133.49
water		59.18	74.70
rms deviations
bond lengths (Å)	0.004	0.004	0.004
bond angles (deg)	0.92	0.91	0.64

^{^a}

Values in parentheses are for the highest-resolution shell.

The transporter adopts the same basic fold as previously published bacterial POTs: (6−13) It is built from two six-helical MFS domains separated by two additional transmembrane helices (TMs) named HA and HB (Figure 1a). MFS transporters can adopt inward facing, occluded, or outward facing conformational states. (14) Here, the transporter is in the inward open form, which is stabilized by the conformation-selective binding of N00 across the interface of the two MFS domains on the periplasmic side (Supplementary Figure 3).

The valganciclovir-bound DtpA-N00 structure is highly similar to the ligand-free structures, with an rmsd of 0.33 Å over 3809 atoms (glycine buffer condition) and 0.42 Å over 3869 atoms (MES buffer condition). Clear electron density could be observed for the bound valganciclovir molecule (Figure 2a). Valganciclovir has two chiral centers: one at the Cα of l-valine and the other at the C2 position (the latter is highlighted with a red star in Figure 1b). It is synthesized as a mix of two diastereomers that only differ at the C2 position. Both diastereomers can be transported by hPepT1 (human peptide transporter 1). (15) It was not possible to distinguish between the two diastereomers in the electron density map of DtpA (Figure 2b).

Figure 2. Binding mode of valganciclovir in the crystal structure of the DtpA-N00-valganciclovir complex. (a) (Left) Valganciclovir is shown in sticks in the ligand-binding site of DtpA, which is illustrated as a surface model (MFS domains are colored as in Figure 1). Nanobody is omitted for clarity. (Right) Omit map for valganciclovir contoured at the 3 – σ level. (b) 2F_o– F_c electron-density map contoured at 1 – σ of the refined DtpA-N00 structure bound to two diastereomers of valganciclovir. All further figures are prepared with diastereomer-1 (left). (c) Interactions of valganciclovir in the binding site. Same coloring as in other panels, but the cartoon representation is at 50% transparency. Potential hydrogen bonds ≤ 3.4 Å are shown as dashes.

The valganciclovir-bound structure reveals an unexpected binding mode for the prodrug. The ligand-binding site of POTs has been previously characterized from structures of different transporters bound to di/tripeptides (9,10,16,17) or the phosphonodipeptide alafosfalin. (7,18) Since valganciclovir has a length similar to tripeptides, it has been suggested that the N-terminal valine residue of the prodrug would mimic the N-terminal residue of a di/tripeptide when bound to the transporter. (19) However, in our DtpA-N00-valganciclovir structure, it is the guanine moiety and not the valine residue that occupies the position corresponding to the N-terminal residue of a peptide substrate. The guanine base is coordinated by Y38, Y156, N160, N325, and E396, which are equivalent to the residues that have previously been observed to bind the N-terminal residues of peptide substrates (16,18) (Figure 2c, Supplementary Figure 4).

The N-terminal valine residue of valganciclovir extends instead into a pocket formed by TM7 and an intrahelical loop in TM10 (I399-SGLG-L404). Here it forms van der Waals interactions with F289 from TM7 as well as with I399 and L402 from the TM10 loop (Figure 2c). In MFS transporters, it is not uncommon to find intrahelical loops in cavity-lining helices. (14) However, among POTs, a loop within TM10 has previously only been observed in PepT_So2 (peptide transporter from bacterial species Shewanella oneidensis). (18) Here TM7 and the loop within TM10 line the pocket that accommodates the side chains in position two of di- and tripeptides, i.e., “pocket 2” (P2). (10) In PepT_St (peptide transporter from bacterial species Streptococcus thermophilus), which has an uninterrupted TM10, P2 is instead formed by TM2, TM7, and TM11 (9) and is located in a partially overlapping position that is closer to the center of the cavity.

Compared to POT structures with an uninterrupted TM10, the periplasmic half of the helix superimposes well whereas the cytoplasmic half is shifted considerably, which in turn causes TM11 to be pushed away from the binding cavity (Figure 3). For example, in an overlay of PepT_St from S. thermophilus on DtpA, the distance between L404 Cα in the cytoplasmic half of TM10 in DtpA and the equivalent L408 Cα atom in PepT_St is 6.1 Å and the distance between F424 Cα in TM11 of DtpA and the equivalent F428 Cα atom in PepT_St is 6.8 Å (Figure 3a). As a result, the binding cavity is significantly enlarged in POTs with a split TM10 (Figure 3b). Indeed, in comparison to other POT transporters with known structures, the binding site of DtpA is the largest (Figure 3b).

Figure 3. Structural comparison of TM10 and TM11 in DtpA and other known POTs. (a) Structural comparison of the TM10-TM11 region of DtpA (orange) and PepT_St (pdb id 5oxn in gray). Loop within TM10 of DtpA (d1) shifts the C-terminal part of TM10 and displaces the N-terminal part of TM11 by 6.8 Å (d2) relative to the position in PepT_St. Equivalent displacement is not found in PepT_So (pdb id 4uvm in magenta), GkPOT (pdb id 4ikv in light green), DtpD (pdb id 4q65 in cyan), YePepT (pdb id 4w6v in blue), PepT_Xc (pdb id 6ei3 in light pink), or PepT_Sh (pdb id 6exs in wheat) but only in DtpA and PepT_So2 (pdb id 4tph in green). Valganciclovir is shown in yellow. (b) Cavity volume around the Cα atom of the valine moiety in valganciclovir, which is intruding into the space created by the TM10 intrahelical loop, was calculated with a radius of 4 and 7 Å using POVME 2.0 (20,21) for all published POT structures.

We conclude that DtpA binds valganciclovir in a particularly large binding cavity by accommodating its guanine base in a site that in other POTs generally engages the N-terminus of peptides and inserting its valine residue into a pocket, which is largely equivalent to the P2 peptide side chain binding pocket of PepT_So2.

To improve our understanding of DtpA and verify its substrate preferences toward peptides and drugs of different size, we screened a ligand library including 18 dipeptides, 10 tripeptides, and 7 drugs. The substrate specificity of DtpA has so far only been characterized in the whole cell context, mostly using a competition assay with the fluorophore-conjugated dipeptide AK-AMCA (β-Ala-Lys coupled to the fluorescent (7-amino-4-methylcoumarin-3-yl)acetic acid), where the reduction of the uptake of the fluorescent molecule into the cell is measured in the presence of increasing amounts of di/tripeptides or drugs. (4) Here, in addition to this in vivo AK-AMCA competition assay, we performed an in vitro thermal shift assay using differential scanning fluorimetry (DSF) (Figure 4a, Supplementary Table 1). The results from both assays are in good agreement. Ligands that are more stabilizing, as determined by DSF, also show stronger inhibition of AK-AMCA uptake (Figure 4b, Supplementary Figure 5, and Supplementary Table 1).

Figure 4. Functional characterization of DtpA. (a) (Left) Thermal stability of DtpA screened with the ligand library at 5 mM concentration. (Right) Notched box plot to compare the thermal stability of DtpA in the presence of di- and tripeptides (W = 46, n₁ = 10, n₂ = 18, P = 0.035, two tailed). (b) (Left) AK-AMCA uptake with the same ligand library at 0.5 mM. (Right) Notched box plot to compare AK-AMCA competition uptake for di- and tripeptides. Results here were subtracted from 100% to ease comparison with the thermal stability results (W = 37, n₁ = 10, n₂ = 18, P = 0.005, two tailed). (c) Binding affinity curve of DtpA with the tripeptide LLA in black and DtpA-N00 with LLA in red. (d) AK-AMCA uptake of DtpA mutants and cells coexpressing DtpA and N00.

With regard to binding of peptides, a U-test performed on the DSF results indicates that binding of tripeptides increases the thermal stability of DtpA (median = 7.1 °C) significantly more than binding of dipeptides (median = 2.4 °C) (W = 46, n₁ = 10, n₂ = 18, P = 0.035, two tailed; Figure 4a, right). In line with this, the increase of median values for the AK-AMCA competition is significantly higher for tripeptides (median = 68%) compared to dipeptides (median = 53%) (W = 37, n₁ = 10, n₂ = 18, P = 0.005, two tailed; Figure 4b, right). The significant difference of binding and transport between di- and tripeptides was further substantiated by a principal component analysis of the physicochemical properties of the tested peptide library. This analysis showed that the molar volume of a peptide ligand is indeed the most strongly contributing variable (Supplementary Figure 6). Finally, quantitative binding affinity measurements were carried out using microscale thermophoresis (MST) for a subset of these ligands, all showing binding affinities in the low to medium micromolar range (Supplementary Figure 7 and Supplementary Table 1). Notably, the highest binding affinity measured by MST was obtained for the tripeptide LLA (K_d = 58 ± 10 μM; Figure 4c), which also induced the highest thermal stabilization, as determined by DSF, and the strongest inhibitory effect on AK-AMCA uptake of all tested ligands (Figure 4a and 4b and Supplementary Table 1). Taken together, our results indicate that DtpA preferably binds and likely transports tripeptides over dipeptides. This is in contrast to most other POTs with known structures, which show preferences toward dipeptides. (9) However, PepT_So2, (10) which shares the presence of an intrahelical loop in TM10 with DtpA, is an exception to this rule (Figure 3a), supporting the notion that a split TM10 and the associated enlargement of the binding cavity may be an adaptation for improving or enabling binding and transport of larger substrates.

From all drugs tested, only valacyclovir and valganciclovir showed a thermal stabilization effect on DtpA and prominent AK-AMCA competition (Figure 4a and 4b). Remarkably, these two drugs bind as tightly to the protein as the best binding tripeptides. The affinities measured using MST were 60 ± 13 μM for valacyclovir and 76 ± 16 μM for valganciclovir (Supplementary Table 1), similar to the K_d of 49 ± 0.3 μM recently measured for the binding of valacyclovir to human PepT1. (22)

Next, we measured the effect of N00 on peptide uptake and affinity in vivo and in vitro. AK-AMCA uptake in cells overexpressing DtpA together with N00 was strongly reduced (Figure 4d). In addition, DtpA modified with a site-specific unnatural amino acid could be cross-linked to N00 upon UV irradiation (Supplementary Figure 8) in lysates, suggesting that N00 stabilizes the protein in the inward open conformation not only in the crystal and in detergent-solubilized form but also in the lipid membrane. Differences in binding affinity upon addition of N00 were typically small (about 2-fold or less), but an increase in affinity of 4–5-fold could be observed for two of the peptides, LA and AL (Supplementary Figure 7 and Supplementary Table 1), indicating that substrate binding affinities may in some cases vary between the inward and the outward open states.

To confirm the location of the ligand-binding site of DtpA by functional assays, five residues (Y38, Y71, K130, N160, and I399) were mutated to alanine. The correct folding of the mutants was confirmed by binding of the conformational nanobody N00 (Supplementary Figure 9a). Binding affinities of these mutants for selected di/tripeptides and the two drugs were strongly reduced relative to wild type (Supplementary Figure 9b and Supplementary Table 2), and AK-AMCA uptake was abolished (Figure 4d). Two additional binding site mutants (Y156A and F289L) that were characterized previously were also found to abrogate AK-AMCA uptake. (23) We therefore conclude that the mutational and structural data are in good agreement.

Taking advantage of the high sequence conservation in the binding site (Supplementary Figure 10), a homology model of human PepT1 (hPepT1) in complex with valganciclovir was built based on the DtpA structure, covering the whole sequence except for the extracellular domain (ECD) and the linker between the two MFS domains (Figure 5). All residues involved in valganciclovir binding in DtpA are observed in corresponding positions in hPepT1. Remarkably, the residues coordinating the guanine base of valganciclovir are fully conserved among DtpA (Y38, Y156, N160, E396) and hPepT1 (Y31, Y167, N171, E595) (Supplementary Figure 11). The residues interacting with the N-terminal valine residue form similar hydrophobic pockets in both proteins.

Figure 5. Ligand-binding site of the human PepT1 model with valganciclovir. hPepT1 model is colored in cyan (N-terminal MFS domain) and in pink (C-terminal domain). Binding site residues are shown in sticks and labeled, and potential hydrogen bonds are shown as dashes (distances ≤ 3.4 Å).

TM5, TM7, and TM10 of hPepT1 have previously been systematically studied by mutations. Here, Y167, N171, and S174 on TM5, (24) F293 and F297 on TM7, (25) and E595 and Y598 on TM10 (26) were suggested to play a role in ligand coordination (Supplementary Figure 12). Indeed, all of these residues form part of the ligand-binding site in our model, and most are in close proximity to valganciclovir. Furthermore, high solvent accessibility was observed for residues T601-E604, (26) in line with these residues forming an intrahelical loop in TM10, as suggested by the hPepT1 model. Thus, this model explains substrate-binding similarities between hPepT1 and DtpA and, in the absence of a crystal structure of hPepT1, can serve as a model for analyzing di/tripeptide and drug interactions.

Concluding Remarks

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We determined the ligand-free and valganciclovir-bound structures of DtpA, a bacterial SLC15 homologue with high similarity to hPepT1, not only in terms of sequence but also regarding substrate specificity and functional properties relating to drug transport. We find that DtpA, in contrast to most characterized bacterial POTs to date, prefers to bind and transport tripeptides over dipeptides, which appears to relate to the presence of a characteristic intrahelical loop in TM10. The structure of DtpA in complex with valganciclovir shows that the prodrug binds in a similar position to di/tripeptides but with the guanine base instead of the valine residue mimicking the N-terminal residue of a di/tripeptide in the ligand-binding site. On the basis of homology modeling and a survey of published mutational data, we find that this binding mode most likely also applies to hPepT1. These novel insights into the structure and substrate binding of DtpA may facilitate future development of prodrugs with improved absorption rates for hPepT1, thereby lowering the pharmacologically effective dose and reducing side effects.

Methods

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Chemicals

Terrific broth (TB) was from Melford, isopropyl-β-d-thiogalactopyranoside (IPTG) and 1,4-dithiothreitol (DTT) were from Roth, DNase I was from AppliChem, complete EDTA-free protease inhibitor cocktail was from Roche, n-dodecyl-β-d-maltopyranoside (DDM) was from Anatrace, and crystallization reagents were from Molecular Dimensions. Amino acids, di/tripeptides, and drugs were obtained from Sigma-Aldrich, Bachem, and Fluka. The unnatural amino acid H-p-Bz-Phe-OH (pBpF) was purchased from Bachem.

Gene Construction, Protein Expression, and Purification of DtpA

The full-length dtpA (Uniprot ID: P77304) gene was amplified from the Escherichia coli genome and cloned into the ptH27 vector. (27) The construct has an N- and C-terminal His₆-tag and a TEV cleavage site after the N-terminal tag. Point mutations were generated by the blunt-end PCR method. DtpA, wild type (WT) and mutants, were expressed and purified as previously described. (28) Briefly, DtpA was overexpressed in E. coli C41(DE3) cells in TB medium. The cell pellet was resuspended in lysis buffer (20 mM NaP, pH 7.5, 300 mM NaCl, 5% glycerol, 15 mM imidazole) with 5 units/ml DNase I, 1 tablet of protease inhibitor/100 mL buffer, 1 mg/mL lysozyme, and 0.5 mM TCEP followed by cell lysis with an emulsifier (EmulsiFlex-C3, Avestin, three passages). The lysate was centrifuged 12 min at 10 000g and the supernatant centrifuged for 50 min at 95 000g (Optima XE-90, Beckman Coulter). The pellet containing the membrane fraction was solubilized in 1% DDM (Anatrace, sol-grade) for 1 h. The sample was centrifuged for 50 min at 70 000g and the supernatant applied to Ni-IMAC beads (ThermoFischer). After 45 min incubation on a rotating wheel, the suspension was transferred to a gravity column. Following two wash steps with lysis buffer supplied with 0.5 mM TCEP, 0.03% DDM, and 40 mM imidazole, DtpA was eluted with lysis buffer containing 0.5 mM TCEP, 0.03% DDM, and 300 mM imidazole. TEV cleavage was performed overnight during dialysis in gel filtration buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM DTT, 0.5% glycerol, and 0.03% DDM), and the protein was further purified on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare). All steps following cell lysis were performed at 4 °C. The protein was concentrated to 11 mg/mL using a 50 kDa cutoff concentrator (Corning Spin-X UF concentrators), flash frozen, and stored at −80 °C until further use.

Selection, Expression, and Purification of Nanobodies Against DtpA

For the generation of DtpA-specific nanobodies, two noninbred llamas were injected six times at weekly intervals with a mixture of 94 different proteins (50 μg of each antigen weekly). After 6 weeks of immunization, two separate phage display libraries were constructed, one from each animal, in the pMESy2 vector, which is a derivative of pMESy4 that contain a C-terminal EPEA-tag for affinity purification. After pooling both libraries, nanobodies were selected against individual antigens in two rounds of parallel panning in 96-well plates containing one immobilized antigen in each well. After two selection rounds on DtpA, 70 clones were picked for sequence analysis, 22 clones encoded antigen-specific nanobodies as tested in ELISA, grouping them in 16 different sequence families. A nanobody family is defined as a group of nanobodies with a high similarity in their CDR3 sequence (identical length and >80% sequence identity). Nanobodies from the same family derive from the same B-cell lineage and likely bind to the same epitope on the target. Immunizations, library construction, selection by panning, and nanobody characterization were performed according to standard procedures. (29) Four nanobodies were further characterized.

The nanobodies were expressed in E. coli WK6 cells and purified following standard procedures. Specifically, the cell pellet was resuspended in TES buffer (0.2 M TRIS, pH 8, 0.5 mM EDTA, 0.5 M sucrose) supplemented with one protease inhibitor tablet (Roche). Osmotic shock was performed by the addition of diluted TES buffer to release the periplasmic proteins. The solution was first centrifuged for 20 min at 10 000g and additionally for 30 min at 142 000g. The supernatant was applied to CaptureSelect beads (Thermo Fisher Scientific), which were equilibrated with wash buffer (20 mM NaP, pH 7.5, 20 mM NaCl). After three column volumes of washing, the nanobody was eluted with 20 mM HEPES, pH 7.5, 1.5 M MgCl₂. The nanobodies were further purified on a HiLoad 16/600 Superdex 75 pg column in 20 mM HEPES, pH 7.5, 150 mM NaCl, 5% glycerol, concentrated with a 5 kDa cutoff concentrator to 13 mg/mL, flash frozen, and stored at −80 °C until further use.

Analytical Gel Filtration Assay

The DtpA-nanobody complex formation was evaluated with an analytical gel filtration chromatography setup using a Superdex 200 5/150 GL home-packed column (GE Healthcare) attached to a 1260 Infinity liquid chromatography system (Agilent technologies). The size exclusion profiles of the samples containing both DtpA and nanobodies were compared to the separate profiles for DtpA and the nanobody (at the same concentrations). The samples were run in 20 mM NaP, pH 7.5, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP, and 0.03% DDM. The concentration of DtpA was 0.2 mg/mL. All samples were run in duplicates at 10 °C following the UV absorption and the fluorescence (excitation at 280 nm and emission at 350 nm). The data analysis was performed in GraphPad Prism 5.0.

Evaluation of Conformational or Linear Epitope Binding

To determine whether the selected nanobodies are conformational or linear epitope binders, Western blot analysis was used (the principle of this approach is illustrated in Supplementary Figure 1a). SDS-PAGE using a 4–12% Bis-TRIS gel (ThermoFisher) under reducing conditions was performed with DtpA using a total amount of 0.3 and 0.03 μg per lane. On the same gel a positive control (N00, amount 15 μg) and a negative control (DtpC, another E. coli POT member with 0.3 and 0.03 μg) were included. The protein was transferred to a PVDF membrane (Biorad). One percent bovine serum albumin in TBS-T (TRIS buffer with Tween20, from Sigma) was used for blocking; TBS-T was also used as washing buffer. The membrane was incubated for 1 h with one of the nanobodies N87, N89, N93, or N00 containing an EPEA tag (1 μg/mL nanobody diluted in TBS-T buffer). Anti-EPEA antibody coupled to biotin (ThermoFisher) was used as primary and streptavidin coupled to horseradish peroxidase (ThermoFisher) as secondary antibody. The blot was developed using Super Signal West Pico Substrate (ThermoFisher) and Super Signal West Femto Substrate (ThermoFisher) in a 1:10 ratio. The image was acquired with a ChemiDoc MP device (BioRad).

Cross-Linking of DtpA and N00 in the Lipid Bilayer

In a cross-linking experiment we investigated if N00 could bind to DtpA in the native membrane. For this experiment two mutants were generated with an UV-inducible cross-linker in different positions. One mutant (G53TAG) was at the proximity of the N00 binding site on the periplasmic side of the transporter, and the second mutant (F265TAG) was on the cytoplasmic side of the transporter as a control. The amber suppression plasmid pEvol-pBpF and the DtpA expression plasmid carrying the amber mutation were cotransformed and overexpressed in the E. coli stain C41(DE3). The cross-linking protocol from Farrell et al. was followed. (30) Shortly, DtpA mutants were overexpressed in medium containing 1 mM pBpF and the lysed cells were incubated with N00. Cross-linking was performed with UV light (365 nm, 60 min), and the DtpA mutant was purified as described above. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4–12% Bis-TRIS gel (ThermoFisher) was performed and transferred on a PVDF membrane (Biorad). Two percent bovine serum albumin in TBS-T (TRIS buffer with Tween20, from Sigma) was used for blocking, TBS-T was used as washing buffer. The membrane was incubated with HisProbe-HRP conjugate antibody (ThermoFisher) for 1 h. The blot was developed using Super Signal West Pico Substrate (ThermoFisher) and Super Signal West Femto Substrate (ThermoFisher) in a 1:10 ratio. The image was acquired with a ChemiDoc MP device (BioRad).

Crystallization and Structure Determination

DtpA and N00 were mixed in 1:1.2 molar ratio 1 h prior to crystallization and incubated at 4 °C. The final concentration of DtpA was 8.5 mg/mL. Crystallization plates were prepared with an automated liquid handler (mosquito, TTP Labtech) using the sitting drop vapor diffusion technique with a final drop volume of 300 nL (at 1:1, 1:2 and 2:1 (v/v) ratios of protein to precipitant). The initial crystals of the ligand-free structure were obtained from the MemGold2 crystallization screen (Molecular Dimensions) at room temperature. After several rounds of optimization, the best diffracting crystals grew in 0.02 M magnesium chloride, 0.1 M MES at pH 6.5, and 32% PEG 600. Diffraction data were collected at the MASSIF-1 (ID30A-1) beamline at ESRF (Grenoble, France) (31) using the fixed 0.969 Å wavelength at 100 K temperature. The data were processed using the XDS package. (32) The space group was identified as P2₁2₁2₁, and the resolution limit was at 3.29 Å. Initial phases were obtained by molecular replacement using DtpD (PDB ID 4Q65) and a nanobody structure as search models in Phaser (33) as part of PHENIX. (34) The model was further built manually in Coot (35) and refined in PHENIX in iterative cycles. The Ramachandran statistics for the final model are 97.3% for favored regions, 2.7% for allowed regions, and 0.0% for outliers. In the case of the DtpA-N00 structure bound to valganciclovir, the drug was added to a DtpA solution in powder form (estimated concentration of 20 mM) and N00 was added as described above. The workflow for structure determination of the drug-bound complex was the same as for the ligand-free form, but the best diffracting crystals grew in the following condition: 0.1 M glycine, pH 9.0, 35% PEG 400, 0.15 M CaCl₂, and 0.02% Anapoe-C₁₂E₁₀. The data set was collected at beamline P13 operated by EMBL Hamburg at the PETRA III storage ring (DESY, Hamburg, Germany) (36) at 0.966 Å wavelength and 100 K temperature. The ligand-free structure was used as a search model for molecular replacement. The Ramachandran statistics were the following: 98.1% favored, 1.9% allowed, and 0.0% for outliers. A second ligand-free structure was determined from crystals derived in a similar condition as the complex structure to validate that the additional density originates from the added compound and not from a buffer or precipitant molecule. Crystals for the second ligand-free form grew in in 0.1 M glycine, pH 9.0, 0.15 M CaCl₂, and 35% PEG 400, and the data were collected at beamline P14 at PETRA III at 0.976 Å wavelength and 100 K temperature and processed as described for the valganciclovir-bound structure. The Ramachandran statistics for the final model are 96.6% for favored regions, 3.4% for allowed regions, and 0.0% for outliers. Structure figures were generated using PyMOL. (37)

Analysis of the Binding Site Volume

The cavity introduced by the TM10 intrahelical loop and the shift of TM11 in DtpA was investigated by comparing the cavity volumes of all available POT structures. The volume calculation was performed with POVME 2.0 (21) using the default parameters. All structures were overlaid, and the coordinates of the Cα atom of the valine moiety in valganciclovir were selected as the center. The volume of the binding site based on a 4 and 7 Å radius sphere was calculated for the following POT structures: DtpA (6GS4), PepT_So2 (4LEP), DtpD (4Q65), YePepT (4W6V), GkPOT (4IKV), PepT_Xc (6EI3), PepT_So (4UVM), PepT_St (5OXN), and PepT_Sh (6EXS). The results were analyzed in GraphPad Prism 5.0 (GraphPad Software, CA, USA).

Thermal Stability Assay

The differential scanning fluorimetry (DSF) method was used to follow the thermal unfolding event of DtpA with a Prometheus NT.48 device (NanoTemper Technologies, Munich, Germany). Here, the fluorescence at 330 and 350 nm is recorded over a temperature gradient scan. The temperature at the transition point of the fluorescence ratio from 330 to 350 nm corresponds to the melting temperature (T_m). The shift of T_m in the presence of a nanobody or a ligand is interpreted as potential binding. DtpA was diluted to 0.5 mg/mL with assay buffer (100 mM HEPES, pH 7.5, 150 mM NaCl, 0.03% DDM). All ligand lyophilizates were dissolved in water to a final concentration of 50 mM. An 18 μL amount of DtpA at 0.5 mg/mL was mixed with 2 μL of 50 mM ligand/substrate (5 mM final concentration). In the case of the nanobodies, the same concentration of DtpA was mixed with a 1:1.2 molar ratio of the tested nanobody. Samples were incubated for 10 min at room temperature before loading them with standard capillaries into the Prometheus device. The excitation power was set between 15% and 25%, and the tested temperature range was from 20 to 85 °C. All measurements were done in triplicate, and the results were analyzed with Excel (Microsoft) and GraphPad Prism 5.0 (GraphPad Software, CA, USA).

Binding Affinity Assay

The binding affinity of DtpA and the di/tripeptides were measured with a label-free microscale thermophoresis device Monolith (NanoTemper Technologies). However, due to the intrinsic signal from valganciclovir and valacyclovir another approach was taken for these drugs. DtpA was labeled on the His₆-tag with RED-tris-NTA dye (NanoTemper), and the binding affinity was measured with microscale thermophoresis following the RED-dye signal. To compare the results from unlabeled and labeled DtpA, the binding affinity of the tripeptide AFA was measured in both systems.

For the measurements on unlabeled DtpA, the protein was diluted to 0.5 μM with assay buffer (100 mM HEPES, pH 7.5, 150 mM NaCl, 0.03% DDM). A dilution series of the substrate was also prepared with the same buffer. DtpA was then mixed with the substrate dilution series and incubated for 10 min at room temperature before loading the samples in standard capillaries to the MST LabelFree device (NanoTemper Technologies, Munich, Germany). For the measurements with the DtpA-N00 complex, the concentration of DtpA was kept constant and a 1:1.2 molar ratio of N00 was mixed 1 h prior to the measurements. As a negative control, the binding affinity of N00 alone, at 2 μM concentration, to selected peptides was measured under the same conditions. The settings were 20% LED and 20% MST power. For the measurements with valganciclovir and valacyclovir, DtpA was diluted to 1.6 μM and labeled with RED-tris-NTA dye according to the supplier’s protocol but using the assay buffer in all steps. The MST NT.115 device (NanoTemper Technologies, Munich, Germany) was used with the standard capillaries and 20% LED and 20% MST power settings. All measurements were performed in duplicate, and the results were analyzed with GraphPad Prism 5.0.

In Vivo Uptake and Competition Assay

The in vivo uptake of the β-Ala-Lys peptide coupled to the fluorescent AMCA (7-amino-4-methylcoumarin-3-yl)acetic acid) group (AK-AMCA) was performed as previously described with minor modifications. (4)E. coli C41(DE3) cells containing the DtpA plasmid were grown in 5 mL of LB medium supplemented with 100 μg/mL ampicillin to an OD_600 nm of 0.7. DtpA expression was induced by the addition of 0.2 mM IPTG and followed by an incubation period of 3 h at 37 °C. The cell culture was then centrifuged for 3 min at 3000 rpm and resuspended to a final OD_600 nm of 10 in the assay buffer (100 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM glucose). 40 μL of buffer, 40 μL of cells, 10 μL of AK-AMCA (final concentration 50 μM), and 10 μL of the competing ligand (final concentration 5 or 0.5 mM) were mixed in a 96-well plate and incubated for 20 min at 37 °C. The reaction was stopped by adding 200 μL of ice-cold assay buffer, and the cells were then washed twice with the same buffer. Finally, the cells were resuspended in 200 μL of assay buffer, and the fluorescence was measured in a M1000 microplate reader (TECAN) with excitation at 350 nm and emission at 450 nm. All experiments were performed in triplicate. The results were normalized by the fluorescence value of the control (cells overexpressing DtpA incubated with AK-AMCA) and plotted as AK-AMCA uptake rate percentage. The analysis was done in Excel and GraphPad Prism 5.0.

Principal Component Analysis

The physicochemical properties of di- and tripeptides investigated in this study were obtained from the ChemSpider database (38) using experimental or predicted physicochemical properties (PhysChem Module of the ACD/Laboratories Percepta Platform; Advanced Chemistry Development Inc., Toronto, Canada). A statistical analysis of the data set was performed in the R Statistical Software Environment. (39) An exploratory statistical analysis by principal component analysis (PCA) was carried out using the R package FactoMineR (40) to investigate potential relationships between physicochemical properties of di- and tripeptide ligands and the two experimentally determined biophysical parameters AK-AMCA competition and thermal stability. Results were visualized with factoextra (41) and ggplot2. (42) Prior to the PCA, the data were filtered for zero variance physicochemical properties. A selection of the 10 most highly contributing properties used for PCA were plotted on a biplot together with the supporting experimentally determined parameters AK-AMCA competition rate and thermal stability that were not used for PCA. Additionally, nonparametric statistical testing was performed to compare the experimentally determined biophysical parameters AK-AMCA competition rate and thermal stability for the groups of di- and tripeptides.

Model Building of Human PepT1 (hPepT1, SLC15A1) with Valganciclovir

The homology model of hPepT1 (Uniprot ID P46059) transmembrane domain was constructed using MODELLER (43) based on the DtpA structure as the modeling template. The initial query-template alignment for the homology model was created with the HH-suite. (44) The alignment was subsequently adjusted manually using the Swiss-PdbViewer (45) according to predictions of secondary structure and membrane helices from GeneSilico MetaServer (46) and to minimize distortions around loop regions. The regions including the ECD domain (47) as well as the eukaryotic linker between TM6 and TM7 were excluded from modeling.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b11343.

Nanobody screening for DtpA binding; electron density maps of DtpA; DtpA-N00 binding site; stereoimage of the ligand-binding site with valganciclovir; AK-AMCA uptake and concentration-dependent competition assay with DtpA; principal component analysis of physico-chemical data for di- and tripeptide ligands; binding curves derived from microscale thermophoresis experiment for DtpA and the DtpA-N00 complex; cross-linking of DtpA and N00 in a lipid environment; thermal stability and binding affinity analysis of DtpA mutants; sequence alignment of DtpA and hPepT1; ligand-binding site of DtpA and hPepT1 with valganciclovir; key residues for function in TM5, TM7, and TM10 highlighted in the hPepT1 homology model; thermal stability (T_m), binding affinity (K_d), and AK-AMCA uptake results for DtpA and DtpA-N00; binding affinity results for DtpA mutants; references (PDF)

ja8b11343_si_001.pdf (2.02 MB)

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Author Information

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Corresponding Author
- Christian Löw - Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany; Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, SE-17177 Stockholm, Sweden; http://orcid.org/0000-0003-0764-7483; Email: [email protected]
Authors
- Yonca Ural-Blimke - Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
- Ali Flayhan - Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
- Jan Strauss - Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany; http://orcid.org/0000-0002-6208-791X
- Vasileios Rantos - Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
- Kim Bartels - Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
- Rolf Nielsen - Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany
- Els Pardon - Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels 1050, Belgium; VIB-VUB Center for Structural Biology, VIB, Brussels 1050, Belgium
- Jan Steyaert - Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels 1050, Belgium; VIB-VUB Center for Structural Biology, VIB, Brussels 1050, Belgium
- Jan Kosinski - Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany; Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
- Esben M. Quistgaard - Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607 Hamburg, Germany; Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, SE-17177 Stockholm, Sweden; Department of Molecular Biology and Genetics − DANDRITE, Gustav Wieds Vej 10, Aarhus University, DK-8000 Aarhus C, Denmark
Notes
The authors declare no competing financial interest.
Atomic coordinates and structure factors were deposited in the protein data bank with the following accession numbers: ligand-free DtpA-N00 structure in MES buffer, 6GS1; DtpA-N00 structure in glycine buffer, 6GS7; and DtpA-N00-valganciclovir structure, 6GS4. The homology model of hPepT1 was deposited in the Model Archive (https://www.modelarchive.org/) and the relevant record can be found at https://dx.doi.org/10.5452/ma-ajiw9.

Acknowledgments

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We thank the Sample Preparation and Characterization facility of EMBL Hamburg for support with nanoDSF measurements, MST, and protein crystallization. We would like to thank the group of Thomas R. Schneider at EMBL Hamburg for access to the EMBL beamlines P13 and P14 as well as the staff at ID30A-1/MASSIF-1 at the European Synchrotron Radiation Facility. We acknowledge the support and the use of resources of Instruct-ERIC, part of the European Strategy Forum on Research Infrastructures (ESFRI), and the Research Foundation-Flanders (FWO) for their support to the nanobody discovery. We thank Saif Saifuzzaman and Alison Lundqvist for technical assistance during nanobody discovery. We want to acknowledge Pär Nordlund from the Karolinska Institutet for his support in the beginning of the project. This work was funded by the Horizon2020 program of the European Union (iNEXT grant, project no. 653706), the Swedish Research Council (grant no. 621-2013-5905), and a grant from the German-Israeli Foundation (GIF grant no: G-1288-207.9/2015). J.S. was additionally supported by a fellowship from the EMBL Interdisciplinary Postdoc (EIPOD) programme under Marie Sklodowska-Curie Actions COFUND programme (grant no. 664726).

References

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Role of electrostatic interactions for ligand recognition and specificity of peptide transporters
Boggavarapu Rajendra; Jeckelmann Jean-Marc; Harder Daniel; Ucurum Zohre; Fotiadis Dimitrios
BMC biology (2015), 13 (), 58 ISSN:.
BACKGROUND: Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood. RESULTS: The X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1's substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter. CONCLUSION: This study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.
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Martinez Molledo, M.; Quistgaard, E. M.; Flayhan, A.; Pieprzyk, J.; Löw, C. Multispecific Substrate Recognition in a Proton-Dependent Oligopeptide Transporter. Structure 2018, 26, 467, DOI: 10.1016/j.str.2018.01.005
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Multispecific Substrate Recognition in a Proton-Dependent Oligopeptide Transporter
Martinez Molledo, Maria; Quistgaard, Esben M.; Flayhan, Ali; Pieprzyk, Joanna; Loew, Christian
Structure (Oxford, United Kingdom) (2018), 26 (3), 467-476.e4CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
Proton-dependent oligopeptide transporters (POTs) are important for uptake of dietary di- and tripeptides in many organisms, and in humans are also involved in drug absorption. These transporters accept a wide range of substrates, but the structural basis for how different peptide side chains are accommodated has so far remained obscure. Twenty-eight peptides were screened for binding to PepTSt from Streptococcus thermophilus, and structures were detd. of PepTSt in complex with four physicochem. diverse dipeptides, which bind with millimolar affinity: Ala-Leu, Phe-Ala, Ala-Gln, and Asp-Glu. The structures show that PepTSt can adapt to different peptide side chains through movement of binding site residues and water mols., and that a good fit can be further aided by adjustment of the position of the peptide itself. Finally, structures were also detd. in complex with adventitiously bound HEPES, polyethylene glycol, and phosphate mols., which further underline the adaptability of the binding site.
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Guettou, F.; Quistgaard, E. M.; Raba, M.; Moberg, P.; Löw, C.; Nordlund, P. Selectivity mechanism of a bacterial homolog of the human drug-peptide transporters PepT1 and PepT2. Nat. Struct. Mol. Biol. 2014, 21, 728, DOI: 10.1038/nsmb.2860
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Selectivity mechanism of a bacterial homolog of the human drug-peptide transporters PepT1 and PepT2
Guettou, Fatma; Quistgaard, Esben M.; Raba, Michael; Moberg, Per; Loew, Christian; Nordlund, Paer
Nature Structural & Molecular Biology (2014), 21 (8), 728-731CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
Peptide transporters of the PepT family have key roles in the transport of di- and tripeptides across membranes as well as in the absorption of orally administered drugs in the small intestine. We have detd. structures of a PepT transporter from Shewanella oneidensis (PepTSo2) in complex with three different peptides. The peptides bind in a large cavity lined by residues that are highly conserved in human PepT1 and PepT2. The bound peptides adopt extended conformations with their N termini clamped into a conserved polar pocket. A pos. charged patch allows differential interactions with the C-terminal carboxylates of di- and tripeptides. Here we identify three pockets for peptide side chain interactions, and our binding studies define differential roles of these pockets for the recognition of different subtypes of peptide side chains.
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Quistgaard, E. M.; Martinez Molledo, M.; Löw, C. Structure Determination of a Major Facilitator Peptide Transporter: Inward Facing PepTSt from Streptococcus thermophilus Crystallized in Space Group P3121. PLoS One 2017, 12, e0173126 DOI: 10.1371/journal.pone.0173126
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Structure determination of a major facilitator peptide transporter: inward facing PepTSt from Streptococcus thermophilus crystallized in space group P3121
Quistgaard, Esben M.; Molledo, Maria Martinez; Low, Christian
PLoS One (2017), 12 (3), e0173126/1-e0173126/20CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
Major facilitator superfamily (MFS) peptide transporters (typically referred to as PepT, POT, or PTR transporters) mediate the uptake of di- and tripeptides, and so play an important dietary role in many organisms. In recent years, a better understanding of the mol. basis for this process has emerged, which is in large part due to a steep increase in structural information. Yet, the conformational transitions underlying the transport mechanism are still not fully understood, and addnl. data is therefore needed. Here we report in detail the detergent screening, crystn., exptl. MIRAS phasing, and refinement of the peptide transporter PepTSt from Streptococcus thermophilus. The space group is P3121, and the protein is crystd. in a monomeric inward facing form. The binding site is likely to be somewhat occluded, as the lobe encompassing transmembrane helixes 10 and 11 is markedly bent towards the central pore of the protein, but the extent of this potential occlusion could not be detd. due to disorder at the apex of the lobe. Based on structural comparisons with the 7 previously detd. P212121 and C2221 structures of inward facing PepTSt, the structural flexibility as well as the conformational changes mediating transition between the inward open and inward facing occluded states are discussed. In conclusion, this report improves our understanding of the structure and conformational cycle of PepTSt, and can furthermore serve as a case study, which may aid in supporting future structure detns. of addnl. MFS transporters or other integral membrane proteins.
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Minhas, G. S.; Bawdon, D.; Herman, R.; Rudden, M.; Stone, A. P.; James, A. G.; Thomas, G. H.; Newstead, S. Structural Basis of Malodour Precursor Transport in the Human Axilla. eLife 2018, 7, e34995, DOI: 10.7554/eLife.34995
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Parker, J. L.; Li, C.; Brinth, A.; Wang, Z.; Vogeley, L.; Solcan, N.; Ledderboge-Vucinic, G.; Swanson, J. M. J.; Caffrey, M.; Voth, G. A.; Newstead, S. Proton Movement and Coupling in the POT Family of Peptide Transporters. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 13182, DOI: 10.1073/pnas.1710727114
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Proton movement and coupling in the POT family of peptide transporters
Parker, Joanne L.; Li, Chenghan; Brinth, Allete; Wang, Zhi; Vogeley, Lutz; Solcan, Nicolae; Ledderboge-Vucinic, Gregory; Swanson, Jessica M. J.; Caffrey, Martin; Voth, Gregory A.; Newstead, Simon
Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (50), 13182-13187CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
POT transporters represent an evolutionarily well-conserved family of proton-coupled transport systems in biol. An unusual feature of the family is their ability to couple the transport of chem. diverse ligands to an inwardly directed proton electrochem. gradient. For example, in mammals, fungi, and bacteria they are predominantly peptide transporters, whereas in plants the family has diverged to recognize nitrate, plant defense compds., and hormones. Although recent structural and biochem. studies have identified conserved sites of proton binding, the mechanism through which transport is coupled to proton movement remains enigmatic. Here we show that different POT transporters operate through distinct proton-coupled mechanisms through changes in the extracellular gate. A high-resoln. crystal structure reveals the presence of ordered water mols. within the peptide binding site. Multiscale mol. dynamics simulations confirm proton transport occurs through these waters via Grotthuss shuttling and reveal that proton binding to the extracellular side of the transporter facilitates a reorientation from an inward- to outward-facing state. Together these results demonstrate that within the POT family multiple mechanisms of proton coupling have likely evolved in conjunction with variation of the extracellular gate.
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Yan, N. Structural Biology of the Major Facilitator Superfamily (MFS) Transporters. Annu. Rev. Biophys. 2015, 44, 257, DOI: 10.1146/annurev-biophys-060414-033901
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Sugawara, M.; Huang, W.; Fei, Y. J.; Leibach, F. H.; Ganapathy, V.; Ganapathy, M. E. Transport of Valganciclovir, a Ganciclovir Prodrug, via Peptide Transporters PEPT1 and PEPT2. J. Pharm. Sci. 2000, 89, 781, DOI: 10.1002/(SICI)1520-6017(200006)89:6<781::AID-JPS10>3.0.CO;2-7
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Transport of valganciclovir, a ganciclovir prodrug, via peptide transporters PEPT1 and PEPT2
Sugawara, Mitsuru; Huang, Wei; Fei, You-Jun; Leibach, Frederick H.; Ganapathy, Vadivel; Ganapathy, Malliga E.
Journal of Pharmaceutical Sciences (2000), 89 (6), 781-789CODEN: JPMSAE; ISSN:0022-3549. (Wiley-Liss, Inc.)
In clin. trials, valganciclovir, the valyl ester of ganciclovir, has been shown to enhance the bioavailability of ganciclovir when taken orally by patients with cytomegalovirus infection. We investigated the role of the intestinal peptide transporter PEPT1 in this process by comparing the interaction of ganciclovir and valganciclovir with the transporter in different exptl. systems. We also studied the interaction of these two compds. with the renal peptide transporter PEPT2. In cell culture model systems using Caco-2 cells for PEPT1 and SKPT cells for PEPT2, valganciclovir inhibited glycylsarcosine transport mediated by PEPT1 and PEPT2 with Ki values (inhibition const.) of 1.68 ± 0.30 and 0.043 ± 0.005 mM, resp. The inhibition by valganciclovir was competitive in both cases. Ganciclovir did not interact with either transporter. Similar studies done with cloned PEPT1 and PEPT2 in heterologous expression systems yielded comparable results. The transport of valganciclovir via PEPT1 was investigated directly in PEPT1-expressing Xenopus laevis oocytes with an electrophysiol. approach. Valganciclovir, but not ganciclovir, induced inward currents in PEPT1-expressing oocytes. These results demonstrate that the increased bioavailability of valganciclovir is related to its recognition as a substrate by the intestinal peptide transporter PEPT1. This prodrug is also recognized by the renal peptide transporter PEPT2 with high affinity.
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Lyons, J. A.; Parker, J. L.; Solcan, N.; Brinth, A.; Li, D.; Shah, S. T.; Caffrey, M.; Newstead, S. Structural Basis for Polyspecificity in the POT Family of Proton-Coupled Oligopeptide Transporters. EMBO Rep. 2014, 15, 886, DOI: 10.15252/embr.201338403
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Structural basis for polyspecificity in the POT family of proton-coupled oligopeptide transporters
Lyons, Joseph A.; Parker, Joanne L.; Solcan, Nicolae; Brinth, Alette; Li, Dianfan; Shah, Syed T. A.; Caffrey, Martin; Newstead, Simon
EMBO Reports (2014), 15 (8), 886-893CODEN: ERMEAX; ISSN:1469-221X. (Wiley-VCH Verlag GmbH & Co. KGaA)
An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di- and tripeptide-bound complexes of a bacterial homolog of PepT1, which reveal at least two mechanisms for peptide recognition that operate within a single, centrally located binding site. The dipeptide was orientated laterally in the binding site, whereas the tripeptide revealed an alternative vertical binding mode. The co-crystal structures combined with functional studies reveal that biochem. distinct peptide-binding sites likely operate within the POT/PTR family of proton-coupled symporters and suggest that transport promiscuity has arisen in part through the ability of the binding site to accommodate peptides in multiple orientations for transport.
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Martinez Molledo, M.; Quistgaard, E. M.; Löw, C. Tripeptide Binding in a Proton-Dependent Oligopeptide Transporter. FEBS Lett. 2018, 592, 3239, DOI: 10.1002/1873-3468.13246
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Tripeptide binding in a proton-dependent oligopeptide transporter
Martinez Molledo, Maria; Quistgaard, Esben M.; Loew, Christian
FEBS Letters (2018), 592 (19), 3239-3247CODEN: FEBLAL; ISSN:0014-5793. (Wiley-Blackwell)
Proton-dependent oligopeptide transporters (POTs) are important for the uptake of di-/tripeptides in many organisms and for drug transport in humans. The binding mode of dipeptides has been well described. However, it is still debated how tripeptides are recognized. Here, we show that tripeptides of the sequence Phe-Ala-Xxx bind with similar affinities as dipeptides to the POT transporter from Streptococcus thermophilus (PepTSt). We furthermore detd. a 2.3-Å structure of PepTSt in complex with Phe-Ala-Gln. The phenylalanine and alanine residues of the peptide adopt the same positions as previously obsd. for the Phe-Ala dipeptide, while the glutamine side chain extends into a hitherto uncharacterized pocket. This pocket is adaptable in size and can likely accommodate a wide variety of peptide side chains.
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Guettou, F.; Quistgaard, E. M.; Trésaugues, L.; Moberg, P.; Jegerschöld, C.; Zhu, L.; Jong, A. J. O.; Nordlund, P.; Löw, C. Structural Insights into Substrate Recognition in Proton-Dependent OligoPeptide Transporters. EMBO Rep. 2013, 14, 804, DOI: 10.1038/embor.2013.107
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Structural insights into substrate recognition in proton-dependent oligopeptide transporters
Guettou, Fatma; Quistgaard, Esben M.; Tresaugues, Lionel; Moberg, Per; Jegerschoeld, Caroline; Zhu, Lin; Jong, Agnes Jin Oi; Nordlund, Paer; Loew, Christian
EMBO Reports (2013), 14 (9), 804-810CODEN: ERMEAX; ISSN:1469-221X. (Nature Publishing Group)
Short-chain peptides are transported across membranes through promiscuous proton-dependent oligopeptide transporters (POTs)-a subfamily of the major facilitator superfamily (MFS). The human POTs, PEPT1 and PEPT2, are also involved in the absorption of various drugs in the gut as well as transport to target cells. Here, we present a structure of an oligomeric POT transporter from Shewanella oneidensis (PepTSo2), which was crystd. in the inward open conformation in complex with the peptidomimetic alafosfalin. All ligand-binding residues are highly conserved and the structural insights presented here are therefore likely to also apply to human POTs.
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Samsudin, F.; Parker, J. L.; Sansom, M. S. P.; Newstead, S.; Fowler, P. W. Accurate Prediction of Ligand Affinities for a Proton-Dependent Oligopeptide Transporter. Cell Chem. Biol. 2016, 23, 299, DOI: 10.1016/j.chembiol.2015.11.015
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Accurate Prediction of Ligand Affinities for a Proton-Dependent Oligopeptide Transporter
Samsudin, Firdaus; Parker, Joanne L.; Sansom, Mark S. P.; Newstead, Simon; Fowler, Philip W.
Cell Chemical Biology (2016), 23 (2), 299-309CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
Membrane transporters are crit. modulators of drug pharmacokinetics, efficacy, and safety. One example is the proton-dependent oligopeptide transporter PepT1, also known as SLC15A1, which is responsible for the uptake of the lactam antibiotics and various peptide-based prodrugs. In this study, we modeled the binding of various peptides to a bacterial homolog, PepTSt, and evaluated a range of computational methods for predicting the free energy of binding. Our results show that a hybrid approach (endpoint methods to classify peptides into good and poor binders and a theor. exact method for refinement) is able to accurately predict affinities, which we validated using proteoliposome transport assays. Applying the method to a homol. model of PepT1 suggests that the approach requires a high-quality structure to be accurate. Our study provides a blueprint for extending these computational methodologies to other pharmaceutically important transporter families.
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Durrant, J. D.; de Oliveira, C. A. F.; McCammon, J. A. POVME: an Algorithm for Measuring Binding-Pocket Volumes. J. Mol. Graphics Modell. 2011, 29, 773, DOI: 10.1016/j.jmgm.2010.10.007
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POVME: An algorithm for measuring binding-pocket volumes
Durrant, Jacob D.; de Oliveira, Cesar Augusto F.; McCammon, J. Andrew
Journal of Molecular Graphics & Modelling (2011), 29 (5), 773-776CODEN: JMGMFI; ISSN:1093-3263. (Elsevier Ltd.)
Researchers engaged in computer-aided drug design often wish to measure the vol. of a ligand-binding pocket in order to predict pharmacol. We have recently developed a simple algorithm, called POVME (POcket Vol. MEasurer), for this purpose. POVME is Python implemented, fast, and freely available. To demonstrate its utility, we use the new algorithm to study three members of the matrix-metalloproteinase family of proteins. Despite the structural similarity of these proteins, differences in binding-pocket dynamics are easily identified.
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Durrant, J. D.; Votapka, L.; Sørensen, J.; Amaro, R. E. POVME 2.0: An Enhanced Tool for Determining Pocket Shape and Volume Characteristics. J. Chem. Theory Comput. 2014, 10, 5047, DOI: 10.1021/ct500381c
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POVME 2.0: An Enhanced Tool for Determining Pocket Shape and Volume Characteristics
Durrant, Jacob D.; Votapka, Lane; Soerensen, Jesper; Amaro, Rommie E.
Journal of Chemical Theory and Computation (2014), 10 (11), 5047-5056CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
Anal. of macromol./small-mol. binding pockets can provide important insights into mol. recognition and receptor dynamics. Since its release in 2011, the POVME (POcket Vol. MEasurer) algorithm has been widely adopted as a simple-to-use tool for measuring and characterizing pocket vols. and shapes. The authors here present POVME 2.0, which is an order of magnitude faster, has improved accuracy, includes a graphical user interface, and can produce volumetric d. maps for improved pocket anal. To demonstrate the utility of the algorithm, the authors use it to analyze the binding pocket of RNA editing ligase 1 from the unicellular parasite Trypanosoma brucei, the etiol. agent of African sleeping sickness. The POVME anal. characterizes the full dynamics of a potentially druggable transient binding pocket and so may guide future antitrypanosomal drug-discovery efforts. The authors are hopeful that this new version will be a useful tool for the computational- and medicinal-chemist community.
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Clémençon, B.; Lüscher, B. P.; Hediger, M. A. Establishment of a Novel Microscale Thermophoresis Ligand-Binding Assay for Characterization of SLC Solute Carriers Using Oligopeptide Transporter PepT1 (SLC15 Family) as a Model System. J. Pharmacol. Toxicol. Methods 2018, 92, 67, DOI: 10.1016/j.vascn.2018.03.004
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Establishment of a novel microscale thermophoresis ligand-binding assay for characterization of SLC solute carriers using oligopeptide transporter PepT1 (SLC15 family) as a model system
Clemencon, Benjamin; Luscher, Benjamin P.; Hediger, Matthias A.
Journal of Pharmacological and Toxicological Methods (2018), 92 (), 67-76CODEN: JPTMEZ; ISSN:1056-8719. (Elsevier)
Membrane proteins represent roughly one third of the human proteome and many of them serve as targets of therapeutic drugs. An exception is the SLC solute carrier superfamily with only a handful of approved drugs targeting SLCs. Indeed, for many of the SLCs, the natural transport substrates are still unknown. A major limitation for SLCs has been the difficulty to thoroughly characterize these multimembrane spanning proteins. The intrinsic properties of membrane proteins with alternative hydrophobic and hydrophilic domains lead to instability, making the purifn. tasks even more challenging compared to sol. proteins. This issue also holds true for conventional ligand-binding assays (LBAs) which usually require high-quality, pure and concd. protein samples. Herein, we report a novel binding assay strategy to overcome these issues, taking advantage of a unique combination of yeast expression and microscale thermophoresis (MST). Following yeast overexpression of SLC15A1/PepT1 ortholog from moss Physcomitrella patens, PepTPp, which exhibits remarkable similarity to human PepT1, the approach was validated using dipeptide glycylsarcosine (Gly-Sar) and antiviral prodrug valacyclovir as test substrates. The originality of our approach is based on the comparative anal. of solubilized total membrane prepns. with or without expression of the SLC target of interest, using a yeast strain (S. cerevisiae), in which the corresponding endogenous SLC homolog is depleted. MST is a recently developed technique that takes advantage of the properties of biomols. in soln. to migrate along a temp. gradient. Importantly, this migration is affected by substrate binding. It is being monitored by fluorescence using labeled SLC mols. in the presence of different ligand concns. We herein report a novel MST/yeast-based method to characterize binding of ligands to SLCs without the need for a prior SLC-purifn. step. For validation purposes, we used a close eukaryotic homolog of the human H+-coupled oligopeptide transporter PepT1 (SLC15A1) that mediates uptake of di-tripeptides and peptide-like drugs as a test model. This approach allowed the successful confirmation of the binding of Gly-Sar at the mM range and revealed for the first time the KD of the antiviral prodrug valacyclovir to the PepT1 homolog at around 50 μM. This novel LBA approach is independent of protein purifn. It is suitable for drug discovery as it is upscalable to high throughput compd. screening. It works well for SLC transporters which are underrepresented targets due to their difficulties to study them. Moreover, this approach could make a significant contribution toward "deorphanization" of SLCs, revealing their transport substrates.
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Malle, E.; Zhou, H.; Neuhold, J.; Spitzenberger, B.; Klepsch, F.; Pollak, T.; Bergner, O.; Ecker, G. F.; Stolt-Bergner, P. C. Random Mutagenesis of the Prokaryotic Peptide Transporter YdgR Identifies Potential Periplasmic Gating Residues. J. Biol. Chem. 2011, 286, 23121, DOI: 10.1074/jbc.M111.239657
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Kulkarni, A. A.; Haworth, I. S.; Lee, V. H. L. Transmembrane Segment 5 of the Dipeptide Transporter hPepT1 Forms a Part of the Substrate Translocation Pathway. Biochem. Biophys. Res. Commun. 2003, 306, 177, DOI: 10.1016/S0006-291X(03)00926-4
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Kulkarni, A. A.; Haworth, I. S.; Uchiyama, T.; Lee, V. H. L. Analysis of Transmembrane Segment 7 of the Dipeptide Transporter hPepT1 by Cysteine-scanning Mutagenesis. J. Biol. Chem. 2003, 278, 51833, DOI: 10.1074/jbc.M308356200
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There is no corresponding record for this reference.
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Xu, L.; Haworth, I. S.; Kulkarni, A. A.; Bolger, M. B.; Davies, D. L. Mutagenesis and Cysteine Scanning of Transmembrane Domain 10 of the Human Dipeptide Transporter. Pharm. Res. 2009, 26, 2358, DOI: 10.1007/s11095-009-9952-9
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There is no corresponding record for this reference.
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Woestenenk, E. A.; Hammarström, M.; van den Berg, S.; Härd, T.; Berglund, H. His tag Effect on Solubility of Human Proteins Produced in Escherichia coli: a Comparison between Four Expression Vectors. J. Struct. Funct. Genomics 2004, 5, 217, DOI: 10.1023/B:jsfg.0000031965.37625.0e
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His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors
Woestenenk, Esmeralda A.; Hammarstroem, Martin; van den Berg, Susanne; Haerd, Torleif; Berglund, Helena
Journal of Structural and Functional Genomics (2004), 5 (3), 217-229CODEN: JSFGAW; ISSN:1345-711X. (Kluwer Academic Publishers)
We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at total recombinant protein prodn. levels per g dry cell wt., soly. of the target proteins, and yield of sol. and total protein when purified by immobilized metal ion affinity purifn. It was found that, in general, both N- and C-terminal His6 tags have a noticeable neg. effect on protein soly., but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this neg. effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiol. conditions. The highest protein prodn. levels and yield of purified protein were obtained from a construct with a C-terminal His tag. We also observe a large variation in cell growth rate, which we detd. to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the prodn. level, soly. and tertiary structure content of the target proteins.
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Flayhan, A.; Mertens, H. D. T.; Ural-Blimke, Y.; Martinez Molledo, M.; Svergun, D. I.; Löw, C. Saposin Lipid Nanoparticles: A Highly Versatile and Modular Tool for Membrane Protein Research. Structure 2018, 26, 345, DOI: 10.1016/j.str.2018.01.007
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Saposin Lipid Nanoparticles: A Highly Versatile and Modular Tool for Membrane Protein Research
Flayhan, Ali; Mertens, Haydyn D. T.; Ural-Blimke, Yonca; Martinez Molledo, Maria; Svergun, Dmitri I.; Loew, Christian
Structure (Oxford, United Kingdom) (2018), 26 (2), 345-355.e5CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
Saposin-derived lipid nanoparticles (SapNPs) are a new alternative tool for membrane protein reconstitution. Here we demonstrate the potential and advantages of SapNPs. We show that SapA has the lowest lipid specificity for SapNP formation. These nanoparticles are modular and offer a tunable range of size and compn. depending on the stoichiometric ratio of lipid and saposin components. They are stable and exhibit features typical of lipid-bilayer systems. Our data suggest that SapNPs are versatile and can adapt to membrane proteins of various sizes and architectures. Using SapA and various types of lipids we could reconstitute membrane proteins of different transmembrane cross-sectional areas (from 14 to 56 transmembrane α helixes). SapNP-reconstituted proteins bound their resp. ligands and were more heat stable compared with the detergent-solubilized form. Moreover, SapNPs encircle membrane proteins in a compact way, allowing structural investigations of small membrane proteins in a detergent-free environment using small-angle X-ray scattering.
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Pardon, E.; Laeremans, T.; Triest, S.; Rasmussen, S.; Wohlkönig, A.; Ruf, A.; Muyldermans, S.; Hol, W. G. J.; Kobilka, B. K.; Steyaert, J. A General Protocol for the Generation of Nanobodies for Structural Biology. Nat. Protoc. 2014, 9, 674, DOI: 10.1038/nprot.2014.039
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A general protocol for the generation of Nanobodies for structural biology
Pardon, Els; Laeremans, Toon; Triest, Sarah; Rasmussen, Soren G. F.; Wohlkonig, Alexandre; Ruf, Armin; Muyldermans, Serge; Hol, Wim G. J.; Kobilka, Brian K.; Steyaert, Jan
Nature Protocols (2014), 9 (3), 674-693CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
There is growing interest in using antibodies as auxiliary tools to crystallize proteins. Here we describe a general protocol for the generation of Nanobodies to be used as crystn. chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins and complexes thereof. Our technol. has a competitive advantage over other recombinant crystn. chaperones in that we fully exploit the natural humoral response against native antigens. Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage display of in vivo-matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples illustrate that the outlined procedures are robust, making it possible to solve by Nanobody-assisted X-ray crystallog. in a time span of 6-12 mo.
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Farrell, I. S.; Toroney, R.; Hazen, J. L.; Mehl, R. A.; Chin, J. W. Photo-Cross-Linking Interacting Proteins with a Genetically Encoded Benzophenone. Nat. Methods 2005, 2, 377, DOI: 10.1038/nmeth0505-377
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Photo-cross-linking interacting proteins with a genetically encoded benzophenone
Farrell, Ian S.; Toroney, Rebecca; Hazen, Jennifer L.; Mehl, Ryan A.; Chin, Jason W.
Nature Methods (2005), 2 (5), 377-384CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
A major challenge in understanding the networks of interactions that control cell and organism function is the definition of protein interactions. Solid-phase peptide synthesis has allowed the photo-crosslinkable amino acid p-benzoyl-L-phenylalanine to be site-specifically incorporated into peptide chains, to facilitate the definition of peptide-ligand complexes. The method, however, is limited to the in vitro study of peptides and small proteins. An innovative develpoment allows the incorporation of a site-specific photo-cross-linker into virtually any protein that can be expressed in Escherichia coli, thereby promoting in vivo or in vitro crosslinking of proteins. The method relies on an orthogonal aminoacyl tRNA synthetase-tRnACUA pair that incorporates pBpa at the position encoded by the amber codon (UAG) in any gene transformed into E. coli. The system described in this protocol uses two plasmids: a p15A-based plasmid to express the orthogonal tRNA and synthetase pair (pDULE) and a second plasmid contg. an amber mutant of the gene of interest. To produce the photo-cross-linker-contg. protein, cultures of E. coli carrying both plasmids are grown in the presence of the unnatural amino acid. To photo-cross-link the protein to its binding partner in vivo or in vitro, cells or purified proteins, resp., are exposed to UV light.
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Bowler Matthew W; Nurizzo Didier; Barrett Ray; Beteva Antonia; Bodin Marjolaine; Caserotto Hugo; Delageniere Solange; Dobias Fabian; Flot David; Giraud Thierry; Guichard Nicolas; Guijarro Mattias; Lentini Mario; Leonard Gordon A; McSweeney Sean; Oskarsson Marcus; Schmidt Werner; Snigirev Anatoli; von Stetten David; Surr John; Svensson Olof; Theveneau Pascal; Mueller-Dieckmann Christoph
Journal of synchrotron radiation (2015), 22 (6), 1540-7 ISSN:.
MASSIF-1 (ID30A-1) is an ESRF undulator beamline operating at a fixed wavelength of 0.969 ÅA (12.8 keV) that is dedicated to the completely automatic characterization of and data collection from crystals of biological macromolecules. The first of the ESRF Upgrade MASSIF beamlines to be commissioned, it has been open since September 2014, providing a unique automated data collection service to academic and industrial users. Here, the beamline characteristics and details of the new service are outlined.
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The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic detn. of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the no. of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.
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Phaser is a program for phasing macromol. crystal structures by both mol. replacement and exptl. phasing methods. The novel phasing algorithms implemented in Phaser have been developed using max. likelihood and multivariate statistics. For mol. replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solns. from noise, and for single-wavelength anomalous dispersion exptl. phasing, the new algorithms, which account for correlations between F+ and F-, give better phases (lower mean phase error with respect to the phases given by the refined structure) than those that use mean F and anomalous differences ΔF. One of the design concepts of Phaser was that it be capable of a high degree of automation. To this end, Phaser (written in C++) can be called directly from Python, although it can also be called using traditional CCP4 keyword-style input. Phaser is a platform for future development of improved phasing methods and their release, including source code, to the crystallog. community.
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Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 213-221CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
A review. Macromol. X-ray crystallog. is routinely applied to understand biol. processes at a mol. level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromol. crystallog. structure soln. with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
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CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for x-ray structure soln., structure comparison and anal., and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as 'Coot'.
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Journal of Synchrotron Radiation (2017), 24 (1), 323-332CODEN: JSYRES; ISSN:1600-5775. (International Union of Crystallography)
The macromol. crystallog. P13 beamline is part of the European Mol. Biol. Lab. Integrated Facility for Structural Biol. at PETRA III (DESY, Hamburg, Germany) and has been in user operation since mid-2013. P13 is tunable across the energy range from 4 to 17.5 keV to support crystallog. data acquisition exploiting a wide range of elemental absorption edges for exptl. phase detn. An adaptive Kirkpatrick-Baez focusing system provides an X-ray beam with a high photon flux and tunable focus size to adapt to diverse exptl. situations. Data collections at energies as low as 4 keV (λ = 3.1 Å) are possible due to a beamline design minimizing background and maximizing photon flux particularly at low energy (up to 1011 photons s-1 at 4 keV), a custom calibration of the PILATUS 6M-F detector for use at low energies, and the availability of a helium path. At high energies, the high photon flux (5.4 × 1011 photons s-1 at 17.5 keV) combined with a large area detector mounted on a 2θ arm allows data collection to sub-at. resoln. (0.55 Å). A peak flux of about 8.0 × 1012 photons s-1 is reached at 11 keV. Automated sample mounting is available by means of the robotic sample changer 'MARVIN' with a dewar capacity of 160 samples. In close proximity to the beamline, labs. have been set up for sample prepn. and characterization; a lab. specifically equipped for on-site heavy atom derivatization with a library of more than 150 compds. is available to beamline users.
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The MPI Bioinformatics Toolkit (https://toolkit.tuebingen.mpg.de) is a free, one-stop web service for protein bioinformatic anal. It currently offers 34 interconnected external and inhouse tools, whose functionality covers sequence similarity searching, alignment construction, detection of sequence features, structure prediction, and sequence classification. This breadth has made the Toolkit an important resource for exptl. biol. and for teaching bioinformatic inquiry. Recently, we replaced the first version of the Toolkit, which was released in 2005 and had served around 2.5 million queries, with an entirely new version, focusing on improved features for the comprehensive anal. of proteins, as well as on promoting teaching. For instance, our popular remote homol. detection server, HHpred, now allows pairwise comparison of two sequences or alignments and offers addnl. profile HMMs for several model organisms and domain databases. Here, we introduce the new version of our Toolkit and its application to the anal. of proteins.
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Comparative protein modeling is increasingly gaining interest since it is of great assistance during the rational design of mutagenesis expts. The availability of this method, and the resulting models, has however been restricted by the availability of expensive computer hardware and software. To overcome these limitations, the authors have developed an environment for comparative protein modeling that consists of SWISS-MODEL, a server for automated comparative protein modeling and of the SWISS-PdbViewer, a sequence to structure workbench. The Swiss-PdbViewer not only acts as a client for SWISS-MODEL, but also provides a large selection of structure anal. and display tools. In addn., the authors provide the SWISS-MODEL Repository, a database contg. more than 3500 automatically generated protein models. By making such tools freely available to the scientific community, the authors hope to increase the use of protein structures and models in the process of expt. design.
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Beale, J. H.; Parker, J. L.; Samsudin, F.; Barrett, A. L.; Senan, A.; Bird, L. E.; Scott, D.; Owens, R. J.; Sansom, M. S. P.; Tucker, S. J.; Meredith, D.; Fowler, P. W.; Newstead, S. Crystal Structures of the Extracellular Domain from PepT1 and PepT2 Provide Novel Insights into Mammalian Peptide Transport. Structure 2015, 23, 1889, DOI: 10.1016/j.str.2015.07.016
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Crystal Structures of the Extracellular Domain from PepT1 and PepT2 Provide Novel Insights into Mammalian Peptide Transport
Beale, John H.; Parker, Joanne L.; Samsudin, Firdaus; Barrett, Anne L.; Senan, Anish; Bird, Louise E.; Scott, David; Owens, Raymond J.; Sansom, Mark S. P.; Tucker, Stephen J.; Meredith, David; Fowler, Philip W.; Newstead, Simon
Structure (Oxford, United Kingdom) (2015), 23 (10), 1889-1899CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
Mammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter contg. a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two Ig-like folds connected in tandem, providing structural insight into mammalian peptide transport. Functional and biophys. studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clustering proteolytic activity to the site of peptide transport in eukaryotic cells.

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Cite this: J. Am. Chem. Soc. 2019, 141, 6, 2404–2412

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https://doi.org/10.1021/jacs.8b11343

Published January 15, 2019

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Abstract
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Figure 1
Figure 1. Structure of the peptide transporter-nanobody complex DtpA-N00, and thermal stability with the prodrugs valganciclovir and valacyclovir. (a) Structure of the DtpA-N00 complex (in MES buffer). N-terminal MFS domain is colored in green, C-terminal MFS domain in orange, HA-HB domain in yellow, and N00 in pink. (b) Thermal stabilization effect of valganciclovir and valacyclovir on DtpA, and chemical structure of the prodrugs. Guanine base is highlighted in pale green and the N-terminal valine residue in light orange. Valganciclovir is a mix of two diastereomers with alternative conformations at the chiral center highlighted with a red star.
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Figure 2
Figure 2. Binding mode of valganciclovir in the crystal structure of the DtpA-N00-valganciclovir complex. (a) (Left) Valganciclovir is shown in sticks in the ligand-binding site of DtpA, which is illustrated as a surface model (MFS domains are colored as in Figure 1). Nanobody is omitted for clarity. (Right) Omit map for valganciclovir contoured at the 3 – σ level. (b) 2F_o– F_c electron-density map contoured at 1 – σ of the refined DtpA-N00 structure bound to two diastereomers of valganciclovir. All further figures are prepared with diastereomer-1 (left). (c) Interactions of valganciclovir in the binding site. Same coloring as in other panels, but the cartoon representation is at 50% transparency. Potential hydrogen bonds ≤ 3.4 Å are shown as dashes.
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Figure 3
Figure 3. Structural comparison of TM10 and TM11 in DtpA and other known POTs. (a) Structural comparison of the TM10-TM11 region of DtpA (orange) and PepT_St (pdb id 5oxn in gray). Loop within TM10 of DtpA (d1) shifts the C-terminal part of TM10 and displaces the N-terminal part of TM11 by 6.8 Å (d2) relative to the position in PepT_St. Equivalent displacement is not found in PepT_So (pdb id 4uvm in magenta), GkPOT (pdb id 4ikv in light green), DtpD (pdb id 4q65 in cyan), YePepT (pdb id 4w6v in blue), PepT_Xc (pdb id 6ei3 in light pink), or PepT_Sh (pdb id 6exs in wheat) but only in DtpA and PepT_So2 (pdb id 4tph in green). Valganciclovir is shown in yellow. (b) Cavity volume around the Cα atom of the valine moiety in valganciclovir, which is intruding into the space created by the TM10 intrahelical loop, was calculated with a radius of 4 and 7 Å using POVME 2.0 (20,21) for all published POT structures.
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Figure 4
Figure 4. Functional characterization of DtpA. (a) (Left) Thermal stability of DtpA screened with the ligand library at 5 mM concentration. (Right) Notched box plot to compare the thermal stability of DtpA in the presence of di- and tripeptides (W = 46, n₁ = 10, n₂ = 18, P = 0.035, two tailed). (b) (Left) AK-AMCA uptake with the same ligand library at 0.5 mM. (Right) Notched box plot to compare AK-AMCA competition uptake for di- and tripeptides. Results here were subtracted from 100% to ease comparison with the thermal stability results (W = 37, n₁ = 10, n₂ = 18, P = 0.005, two tailed). (c) Binding affinity curve of DtpA with the tripeptide LLA in black and DtpA-N00 with LLA in red. (d) AK-AMCA uptake of DtpA mutants and cells coexpressing DtpA and N00.
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Figure 5
Figure 5. Ligand-binding site of the human PepT1 model with valganciclovir. hPepT1 model is colored in cyan (N-terminal MFS domain) and in pink (C-terminal domain). Binding site residues are shown in sticks and labeled, and potential hydrogen bonds are shown as dashes (distances ≤ 3.4 Å).
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References
This article references 47 other publications.
1. 1
  Brandsch, M.; Knütter, I.; Bosse-Doenecke, E. Pharmaceutical and Pharmacological Importance of Peptide Transporters. J. Pharm. Pharmacol. 2008, 60, 543, DOI: 10.1211/jpp.60.5.0002
  1
  Pharmaceutical and pharmacological importance of peptide transporters
  Brandsch, Matthias; Knuetter, Ilka; Bosse-Doenecke, Eva
  Journal of Pharmacy and Pharmacology (2008), 60 (5), 543-585CODEN: JPPMAB; ISSN:0022-3573. (Pharmaceutical Press)
  A review. Peptide transport is currently a prominent topic in membrane research. The transport proteins involved are under intense investigation because of their physiol. importance in protein absorption and also because peptide transporters are possible vehicles for drug delivery. Moreover, in many tissues peptide carriers transduce peptidic signals across membranes that are relevant in information processing. The focus of this review is on the pharmaceutical relevance of the human peptide transporters PEPT1 and PEPT2. In addn. to their physiol. substrates, both carriers transport many β-lactam antibiotics, valaciclovir and other drugs and prodrugs because of their sterical resemblance to di- and tripeptides. The primary structure, tissue distribution and substrate specificity of PEPT1 and PEPT2 have been well characterized. However, there is a dearth of knowledge on the substrate binding sites and the three-dimensional structure of these proteins. Until this pivotal information becomes available by X-ray crystallog., the development of new drug substrates relies on classical transport studies combined with mol. modeling. In more than thirty years of research, data on the interaction of well over 700 di- and tripeptides, amino acid and peptide derivs., drugs and prodrugs with peptide transporters have been gathered. The aim of this review is to put the reports on peptide transporter-mediated drug uptake into perspective. We also review the current knowledge on pharmacogenomics and clin. relevance of human peptide transporters. Finally, the reader's attention is drawn to other known or proposed human peptide-transporting proteins.
2. 2
  Jung, D.; Dorr, A. Single-Dose Pharmacokinetics of Valganciclovir in HIV- and CMV-Seropositive Subjects. J. Clin. Pharmacol. 1999, 39, 800, DOI: 10.1177/00912709922008452
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  Single-dose pharmacokinetics of valganciclovir in HIV- and CMV-seropositive subjects
  Jung, Donald; Dorr, Albert
  Journal of Clinical Pharmacology (1999), 39 (8), 800-804CODEN: JCPCBR; ISSN:0091-2700. (Sage Publications)
  As a result of the low oral bioavailability of ganciclovir, a prodrug was developed to improve the bioavailability of ganciclovir. This study was designed to investigate the fasting, single-dose pharmacokinetics as well as the abs. and relative bioavailability of a valine ester prodrug of ganciclovir, valganciclovir, as compared to oral and i.v. ganciclovir in asymptomatic HIV+ and CMV+ subjects. In this open-label, randomized, three-period crossover study, 18 subjects received, in random order, single oral doses of valganciclovir 360 mg and ganciclovir 1000 mg and an i.v. infusion of ganciclovir 5 mg/kg over 1 h. Valganciclovir was rapidly and extensively hydrolyzed to ganciclovir, resulting in significantly greater bioavailability compared to 1000 mg oral ganciclovir (60.9% vs. 5.6%, resp.). Higher peak serum concns. were reached earlier following valganciclovir (ganciclovir [2.98±0.77 μg/mL at 1.0±0.3 h]) than following oral ganciclovir (0.47±0.17 μg/mL and 2.2±1.0 h). Mean total ganciclovir AUCs following oral ganciclovir (1000 mg) and 360 mg valganciclovir (3.8±1.2 and 10.8±1.9 μg-h/mL) were less than that following a std. 5 mg/kg i.v. infusion of ganciclovir (25.1±3.8 μg-h/mL). In summary, valganciclovir is a prodrug with a favorable safety profile with enhanced bioavailability and significantly higher serum concns. of ganciclovir than following oral administration of ganciclovir itself.
3. 3
  Beauchamp, L. M.; Orr, G. F.; de Miranda, P.; Bumette, T.; Krenitsky, T. A. Amino Acid Ester Prodrugs of Acyclovir. Antivir. Chem. Chemother. 1992, 3, 157, DOI: 10.1177/095632029200300305
  3
  Amino acid ester prodrugs of acyclovir
  Beauchamp, L. M.; Orr, G. F.; De Miranda, P.; Burnette, T.; Krenitsky, T. A.
  Antiviral Chemistry & Chemotherapy (1992), 3 (3), 157-64CODEN: ACCHEH; ISSN:0956-3202.
  Eighteen amino acid esters of the antiherpetic drug, acyclovir, were synthesized as potential prodrugs for oral administration. The esters were examd. for in vitro antiviral activity against herpes simplex virus Type 1 (HSV-1). They had less potency than the parent compd. Their efficiencies as prodrugs were evaluated in rats by measuring the urinary recovery of acyclovir. Ten prodrugs produced greater amts. of the parent drug in the urine. The L-amino acid esters were better prodrugs than the corresponding D- or D,L-isomers, suggesting the involvement of a stereoselective transporter. The L-valyl ester, 256U87, was the best prodrug. Sixty-three percent of its administered dose was excreted as acyclovir in the urine, a considerable improvement over acyclovir itself, for which this value was 19%. Since 256U87 was stable in aq. solns., its conversion to acyclovir in vivo was probably enzyme catalyzed. This L-valyl ester prodrug of acyclovir is now undergoing clin. evaluation.
4. 4
  Weitz, D.; Harder, D.; Casagrande, F.; Fotiadis, D.; Obrdlik, P.; Kelety, B.; Daniel, H. Functional and Structural Characterization of a Prokaryotic Peptide Transporter with Features Similar to Mammalian PEPT1. J. Biol. Chem. 2007, 282, 2832, DOI: 10.1074/jbc.M604866200
  There is no corresponding record for this reference.
5. 5
  Prabhala, B. K.; Aduri, N. G.; Iqbal, M.; Rahman, M.; Gajhede, M.; Hansen, P. R.; Mirza, O. Several hPepT1-Transported Drugs are Substrates of the Escherichia coli Proton-Coupled Oligopeptide Transporter YdgR. Res. Microbiol. 2017, 168, 443, DOI: 10.1016/j.resmic.2017.01.005
  5
  Several hPepT1-transported drugs are substrates of the Escherichia coli proton-coupled oligopeptide transporter YdgR
  Prabhala, Bala K.; Aduri, Nanda G.; Iqbal, Mazhar; Rahman, Moazur; Gajhede, Michael; Hansen, Paul R.; Mirza, Osman
  Research in Microbiology (2017), 168 (5), 443-449CODEN: RMCREW; ISSN:0923-2508. (Elsevier Masson SAS)
  Proton-dependent oligopeptide transporters (POTs) are secondary active transporters found in all kingdoms of life. POTs utilize the proton electrochem. gradient for the uptake of nutrient dipeptides and tripeptides. The human POT hPepT1 is known to transport a no. of drugs. As part of ongoing studies on substrate specificities of POTs from Escherichia coli, our aim in this study was to investigate whether bacterial POTs could also transport these drugs. For this, we selected the common orally administered drugs sulpiride, bestatin, valacyclovir, ampicillin and oseltamivir, that are all transported by hPepT1. The transport of these drugs was evaluated using the prototypical POT YdgR from E. coli. The transport studies were pursued through combining cell-based assays with liq. chromatog.-tandem mass spectrometric (LC-MS/MS) anal. These investigations revealed that YdgR from E. coli is able to transport five (sulpiride, bestatin, valacyclovir, ampicillin and oseltamivir) drugs. Furthermore, cells not overexpressing YdgR were also able to transport these drugs in a POT-like manner. Orthologues of YdgR are found in several species in the gut microbiome; hence, our findings could have implications for further understanding about the interaction between gut microbes and orally administered drugs.
6. 6
  Solcan, N.; Kwok, J.; Fowler, P. W.; Cameron, A. D.; Drew, D.; Iwata, S.; Newstead, S. Alternating access mechanism in the POT family of oligopeptide transporters. EMBO J. 2012, 31, 3411, DOI: 10.1038/emboj.2012.157
  6
  Alternating access mechanism in the POT family of oligopeptide transporters
  Solcan, Nicolae; Kwok, Jane; Fowler, Philip W.; Cameron, Alexander D.; Drew, David; Iwata, So; Newstead, Simon
  EMBO Journal (2012), 31 (16), 3411-3421CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)
  Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton-dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3-Å resoln. crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystd. in an inward open conformation the structure identifies a hinge-like movement within the C-terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide-binding site. Our assocd. functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.
7. 7
  Doki, S.; Kato, H. E.; Solcan, N.; Iwaki, M.; Koyama, M.; Hattori, M.; Iwase, N.; Tsukazaki, T.; Sugita, Y.; Kandori, H.; Newstead, S.; Ishitani, R.; Nureki, O. Structural Basis for Dynamic Mechanism of Proton-Coupled Symport by the Peptide Transporter POT. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 11343, DOI: 10.1073/pnas.1301079110
  7
  Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT
  Doki, Shintaro; Kato, Hideaki E.; Solcan, Nicolae; Iwaki, Masayo; Koyama, Michio; Hattori, Motoyuki; Iwase, Norihiko; Tsukazaki, Tomoya; Sugita, Yuji; Kandori, Hideki; Newstead, Simon; Ishitani, Ryuichiro; Nureki, Osamu
  Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (28), 11343-11348, S11343/1-S11343/34CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  Proton-dependent oligopeptide transporters (POTs) are major facilitator superfamily (MFS) proteins that mediate the uptake of peptides and peptide-like mols., using the inwardly directed H+ gradient across the membrane. The human POT family transporter peptide transporter 1 is present in the brush border membrane of the small intestine and is involved in the uptake of nutrient peptides and drug mols. such as β-lactam antibiotics. Although previous studies have provided insight into the overall structure of the POT family transporters, the question of how transport is coupled to both peptide and H+ binding remains unanswered. Here we report the high-resoln. crystal structures of a bacterial POT family transporter, including its complex with a dipeptide analog, alafosfalin. These structures revealed the key mechanistic and functional roles for a conserved glutamate residue (Glu310) in the peptide binding site. Integrated structural, biochem., and computational analyses suggested a mechanism for H+-coupled peptide symport in which protonated Glu310 first binds the carboxyl group of the peptide substrate. The deprotonation of Glu310 in the inward open state triggers the release of the bound peptide toward the intracellular space and salt bridge formation between Glu310 and Arg43 to induce the state transition to the occluded conformation.
8. 8
  Boggavarapu, R.; Jeckelmann, J.-M.; Harder, D.; Ucurum, Z.; Fotiadis, D. Role of Electrostatic Interactions for Ligand Recognition and Specificity of Peptide Transporters. BMC Biol. 2015, 13, 58, DOI: 10.1186/s12915-015-0167-8
  8
  Role of electrostatic interactions for ligand recognition and specificity of peptide transporters
  Boggavarapu Rajendra; Jeckelmann Jean-Marc; Harder Daniel; Ucurum Zohre; Fotiadis Dimitrios
  BMC biology (2015), 13 (), 58 ISSN:.
  BACKGROUND: Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood. RESULTS: The X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1's substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter. CONCLUSION: This study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.
9. 9
  Martinez Molledo, M.; Quistgaard, E. M.; Flayhan, A.; Pieprzyk, J.; Löw, C. Multispecific Substrate Recognition in a Proton-Dependent Oligopeptide Transporter. Structure 2018, 26, 467, DOI: 10.1016/j.str.2018.01.005
  9
  Multispecific Substrate Recognition in a Proton-Dependent Oligopeptide Transporter
  Martinez Molledo, Maria; Quistgaard, Esben M.; Flayhan, Ali; Pieprzyk, Joanna; Loew, Christian
  Structure (Oxford, United Kingdom) (2018), 26 (3), 467-476.e4CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
  Proton-dependent oligopeptide transporters (POTs) are important for uptake of dietary di- and tripeptides in many organisms, and in humans are also involved in drug absorption. These transporters accept a wide range of substrates, but the structural basis for how different peptide side chains are accommodated has so far remained obscure. Twenty-eight peptides were screened for binding to PepTSt from Streptococcus thermophilus, and structures were detd. of PepTSt in complex with four physicochem. diverse dipeptides, which bind with millimolar affinity: Ala-Leu, Phe-Ala, Ala-Gln, and Asp-Glu. The structures show that PepTSt can adapt to different peptide side chains through movement of binding site residues and water mols., and that a good fit can be further aided by adjustment of the position of the peptide itself. Finally, structures were also detd. in complex with adventitiously bound HEPES, polyethylene glycol, and phosphate mols., which further underline the adaptability of the binding site.
10. 10
  Guettou, F.; Quistgaard, E. M.; Raba, M.; Moberg, P.; Löw, C.; Nordlund, P. Selectivity mechanism of a bacterial homolog of the human drug-peptide transporters PepT1 and PepT2. Nat. Struct. Mol. Biol. 2014, 21, 728, DOI: 10.1038/nsmb.2860
  10
  Selectivity mechanism of a bacterial homolog of the human drug-peptide transporters PepT1 and PepT2
  Guettou, Fatma; Quistgaard, Esben M.; Raba, Michael; Moberg, Per; Loew, Christian; Nordlund, Paer
  Nature Structural & Molecular Biology (2014), 21 (8), 728-731CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
  Peptide transporters of the PepT family have key roles in the transport of di- and tripeptides across membranes as well as in the absorption of orally administered drugs in the small intestine. We have detd. structures of a PepT transporter from Shewanella oneidensis (PepTSo2) in complex with three different peptides. The peptides bind in a large cavity lined by residues that are highly conserved in human PepT1 and PepT2. The bound peptides adopt extended conformations with their N termini clamped into a conserved polar pocket. A pos. charged patch allows differential interactions with the C-terminal carboxylates of di- and tripeptides. Here we identify three pockets for peptide side chain interactions, and our binding studies define differential roles of these pockets for the recognition of different subtypes of peptide side chains.
11. 11
  Quistgaard, E. M.; Martinez Molledo, M.; Löw, C. Structure Determination of a Major Facilitator Peptide Transporter: Inward Facing PepTSt from Streptococcus thermophilus Crystallized in Space Group P3121. PLoS One 2017, 12, e0173126 DOI: 10.1371/journal.pone.0173126
  11
  Structure determination of a major facilitator peptide transporter: inward facing PepTSt from Streptococcus thermophilus crystallized in space group P3121
  Quistgaard, Esben M.; Molledo, Maria Martinez; Low, Christian
  PLoS One (2017), 12 (3), e0173126/1-e0173126/20CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
  Major facilitator superfamily (MFS) peptide transporters (typically referred to as PepT, POT, or PTR transporters) mediate the uptake of di- and tripeptides, and so play an important dietary role in many organisms. In recent years, a better understanding of the mol. basis for this process has emerged, which is in large part due to a steep increase in structural information. Yet, the conformational transitions underlying the transport mechanism are still not fully understood, and addnl. data is therefore needed. Here we report in detail the detergent screening, crystn., exptl. MIRAS phasing, and refinement of the peptide transporter PepTSt from Streptococcus thermophilus. The space group is P3121, and the protein is crystd. in a monomeric inward facing form. The binding site is likely to be somewhat occluded, as the lobe encompassing transmembrane helixes 10 and 11 is markedly bent towards the central pore of the protein, but the extent of this potential occlusion could not be detd. due to disorder at the apex of the lobe. Based on structural comparisons with the 7 previously detd. P212121 and C2221 structures of inward facing PepTSt, the structural flexibility as well as the conformational changes mediating transition between the inward open and inward facing occluded states are discussed. In conclusion, this report improves our understanding of the structure and conformational cycle of PepTSt, and can furthermore serve as a case study, which may aid in supporting future structure detns. of addnl. MFS transporters or other integral membrane proteins.
12. 12
  Minhas, G. S.; Bawdon, D.; Herman, R.; Rudden, M.; Stone, A. P.; James, A. G.; Thomas, G. H.; Newstead, S. Structural Basis of Malodour Precursor Transport in the Human Axilla. eLife 2018, 7, e34995, DOI: 10.7554/eLife.34995
  There is no corresponding record for this reference.
13. 13
  Parker, J. L.; Li, C.; Brinth, A.; Wang, Z.; Vogeley, L.; Solcan, N.; Ledderboge-Vucinic, G.; Swanson, J. M. J.; Caffrey, M.; Voth, G. A.; Newstead, S. Proton Movement and Coupling in the POT Family of Peptide Transporters. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 13182, DOI: 10.1073/pnas.1710727114
  13
  Proton movement and coupling in the POT family of peptide transporters
  Parker, Joanne L.; Li, Chenghan; Brinth, Allete; Wang, Zhi; Vogeley, Lutz; Solcan, Nicolae; Ledderboge-Vucinic, Gregory; Swanson, Jessica M. J.; Caffrey, Martin; Voth, Gregory A.; Newstead, Simon
  Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (50), 13182-13187CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  POT transporters represent an evolutionarily well-conserved family of proton-coupled transport systems in biol. An unusual feature of the family is their ability to couple the transport of chem. diverse ligands to an inwardly directed proton electrochem. gradient. For example, in mammals, fungi, and bacteria they are predominantly peptide transporters, whereas in plants the family has diverged to recognize nitrate, plant defense compds., and hormones. Although recent structural and biochem. studies have identified conserved sites of proton binding, the mechanism through which transport is coupled to proton movement remains enigmatic. Here we show that different POT transporters operate through distinct proton-coupled mechanisms through changes in the extracellular gate. A high-resoln. crystal structure reveals the presence of ordered water mols. within the peptide binding site. Multiscale mol. dynamics simulations confirm proton transport occurs through these waters via Grotthuss shuttling and reveal that proton binding to the extracellular side of the transporter facilitates a reorientation from an inward- to outward-facing state. Together these results demonstrate that within the POT family multiple mechanisms of proton coupling have likely evolved in conjunction with variation of the extracellular gate.
14. 14
  Yan, N. Structural Biology of the Major Facilitator Superfamily (MFS) Transporters. Annu. Rev. Biophys. 2015, 44, 257, DOI: 10.1146/annurev-biophys-060414-033901
  There is no corresponding record for this reference.
15. 15
  Sugawara, M.; Huang, W.; Fei, Y. J.; Leibach, F. H.; Ganapathy, V.; Ganapathy, M. E. Transport of Valganciclovir, a Ganciclovir Prodrug, via Peptide Transporters PEPT1 and PEPT2. J. Pharm. Sci. 2000, 89, 781, DOI: 10.1002/(SICI)1520-6017(200006)89:6<781::AID-JPS10>3.0.CO;2-7
  15
  Transport of valganciclovir, a ganciclovir prodrug, via peptide transporters PEPT1 and PEPT2
  Sugawara, Mitsuru; Huang, Wei; Fei, You-Jun; Leibach, Frederick H.; Ganapathy, Vadivel; Ganapathy, Malliga E.
  Journal of Pharmaceutical Sciences (2000), 89 (6), 781-789CODEN: JPMSAE; ISSN:0022-3549. (Wiley-Liss, Inc.)
  In clin. trials, valganciclovir, the valyl ester of ganciclovir, has been shown to enhance the bioavailability of ganciclovir when taken orally by patients with cytomegalovirus infection. We investigated the role of the intestinal peptide transporter PEPT1 in this process by comparing the interaction of ganciclovir and valganciclovir with the transporter in different exptl. systems. We also studied the interaction of these two compds. with the renal peptide transporter PEPT2. In cell culture model systems using Caco-2 cells for PEPT1 and SKPT cells for PEPT2, valganciclovir inhibited glycylsarcosine transport mediated by PEPT1 and PEPT2 with Ki values (inhibition const.) of 1.68 ± 0.30 and 0.043 ± 0.005 mM, resp. The inhibition by valganciclovir was competitive in both cases. Ganciclovir did not interact with either transporter. Similar studies done with cloned PEPT1 and PEPT2 in heterologous expression systems yielded comparable results. The transport of valganciclovir via PEPT1 was investigated directly in PEPT1-expressing Xenopus laevis oocytes with an electrophysiol. approach. Valganciclovir, but not ganciclovir, induced inward currents in PEPT1-expressing oocytes. These results demonstrate that the increased bioavailability of valganciclovir is related to its recognition as a substrate by the intestinal peptide transporter PEPT1. This prodrug is also recognized by the renal peptide transporter PEPT2 with high affinity.
16. 16
  Lyons, J. A.; Parker, J. L.; Solcan, N.; Brinth, A.; Li, D.; Shah, S. T.; Caffrey, M.; Newstead, S. Structural Basis for Polyspecificity in the POT Family of Proton-Coupled Oligopeptide Transporters. EMBO Rep. 2014, 15, 886, DOI: 10.15252/embr.201338403
  16
  Structural basis for polyspecificity in the POT family of proton-coupled oligopeptide transporters
  Lyons, Joseph A.; Parker, Joanne L.; Solcan, Nicolae; Brinth, Alette; Li, Dianfan; Shah, Syed T. A.; Caffrey, Martin; Newstead, Simon
  EMBO Reports (2014), 15 (8), 886-893CODEN: ERMEAX; ISSN:1469-221X. (Wiley-VCH Verlag GmbH & Co. KGaA)
  An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di- and tripeptide-bound complexes of a bacterial homolog of PepT1, which reveal at least two mechanisms for peptide recognition that operate within a single, centrally located binding site. The dipeptide was orientated laterally in the binding site, whereas the tripeptide revealed an alternative vertical binding mode. The co-crystal structures combined with functional studies reveal that biochem. distinct peptide-binding sites likely operate within the POT/PTR family of proton-coupled symporters and suggest that transport promiscuity has arisen in part through the ability of the binding site to accommodate peptides in multiple orientations for transport.
17. 17
  Martinez Molledo, M.; Quistgaard, E. M.; Löw, C. Tripeptide Binding in a Proton-Dependent Oligopeptide Transporter. FEBS Lett. 2018, 592, 3239, DOI: 10.1002/1873-3468.13246
  17
  Tripeptide binding in a proton-dependent oligopeptide transporter
  Martinez Molledo, Maria; Quistgaard, Esben M.; Loew, Christian
  FEBS Letters (2018), 592 (19), 3239-3247CODEN: FEBLAL; ISSN:0014-5793. (Wiley-Blackwell)
  Proton-dependent oligopeptide transporters (POTs) are important for the uptake of di-/tripeptides in many organisms and for drug transport in humans. The binding mode of dipeptides has been well described. However, it is still debated how tripeptides are recognized. Here, we show that tripeptides of the sequence Phe-Ala-Xxx bind with similar affinities as dipeptides to the POT transporter from Streptococcus thermophilus (PepTSt). We furthermore detd. a 2.3-Å structure of PepTSt in complex with Phe-Ala-Gln. The phenylalanine and alanine residues of the peptide adopt the same positions as previously obsd. for the Phe-Ala dipeptide, while the glutamine side chain extends into a hitherto uncharacterized pocket. This pocket is adaptable in size and can likely accommodate a wide variety of peptide side chains.
18. 18
  Guettou, F.; Quistgaard, E. M.; Trésaugues, L.; Moberg, P.; Jegerschöld, C.; Zhu, L.; Jong, A. J. O.; Nordlund, P.; Löw, C. Structural Insights into Substrate Recognition in Proton-Dependent OligoPeptide Transporters. EMBO Rep. 2013, 14, 804, DOI: 10.1038/embor.2013.107
  18
  Structural insights into substrate recognition in proton-dependent oligopeptide transporters
  Guettou, Fatma; Quistgaard, Esben M.; Tresaugues, Lionel; Moberg, Per; Jegerschoeld, Caroline; Zhu, Lin; Jong, Agnes Jin Oi; Nordlund, Paer; Loew, Christian
  EMBO Reports (2013), 14 (9), 804-810CODEN: ERMEAX; ISSN:1469-221X. (Nature Publishing Group)
  Short-chain peptides are transported across membranes through promiscuous proton-dependent oligopeptide transporters (POTs)-a subfamily of the major facilitator superfamily (MFS). The human POTs, PEPT1 and PEPT2, are also involved in the absorption of various drugs in the gut as well as transport to target cells. Here, we present a structure of an oligomeric POT transporter from Shewanella oneidensis (PepTSo2), which was crystd. in the inward open conformation in complex with the peptidomimetic alafosfalin. All ligand-binding residues are highly conserved and the structural insights presented here are therefore likely to also apply to human POTs.
19. 19
  Samsudin, F.; Parker, J. L.; Sansom, M. S. P.; Newstead, S.; Fowler, P. W. Accurate Prediction of Ligand Affinities for a Proton-Dependent Oligopeptide Transporter. Cell Chem. Biol. 2016, 23, 299, DOI: 10.1016/j.chembiol.2015.11.015
  19
  Accurate Prediction of Ligand Affinities for a Proton-Dependent Oligopeptide Transporter
  Samsudin, Firdaus; Parker, Joanne L.; Sansom, Mark S. P.; Newstead, Simon; Fowler, Philip W.
  Cell Chemical Biology (2016), 23 (2), 299-309CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
  Membrane transporters are crit. modulators of drug pharmacokinetics, efficacy, and safety. One example is the proton-dependent oligopeptide transporter PepT1, also known as SLC15A1, which is responsible for the uptake of the lactam antibiotics and various peptide-based prodrugs. In this study, we modeled the binding of various peptides to a bacterial homolog, PepTSt, and evaluated a range of computational methods for predicting the free energy of binding. Our results show that a hybrid approach (endpoint methods to classify peptides into good and poor binders and a theor. exact method for refinement) is able to accurately predict affinities, which we validated using proteoliposome transport assays. Applying the method to a homol. model of PepT1 suggests that the approach requires a high-quality structure to be accurate. Our study provides a blueprint for extending these computational methodologies to other pharmaceutically important transporter families.
20. 20
  Durrant, J. D.; de Oliveira, C. A. F.; McCammon, J. A. POVME: an Algorithm for Measuring Binding-Pocket Volumes. J. Mol. Graphics Modell. 2011, 29, 773, DOI: 10.1016/j.jmgm.2010.10.007
  20
  POVME: An algorithm for measuring binding-pocket volumes
  Durrant, Jacob D.; de Oliveira, Cesar Augusto F.; McCammon, J. Andrew
  Journal of Molecular Graphics & Modelling (2011), 29 (5), 773-776CODEN: JMGMFI; ISSN:1093-3263. (Elsevier Ltd.)
  Researchers engaged in computer-aided drug design often wish to measure the vol. of a ligand-binding pocket in order to predict pharmacol. We have recently developed a simple algorithm, called POVME (POcket Vol. MEasurer), for this purpose. POVME is Python implemented, fast, and freely available. To demonstrate its utility, we use the new algorithm to study three members of the matrix-metalloproteinase family of proteins. Despite the structural similarity of these proteins, differences in binding-pocket dynamics are easily identified.
21. 21
  Durrant, J. D.; Votapka, L.; Sørensen, J.; Amaro, R. E. POVME 2.0: An Enhanced Tool for Determining Pocket Shape and Volume Characteristics. J. Chem. Theory Comput. 2014, 10, 5047, DOI: 10.1021/ct500381c
  21
  POVME 2.0: An Enhanced Tool for Determining Pocket Shape and Volume Characteristics
  Durrant, Jacob D.; Votapka, Lane; Soerensen, Jesper; Amaro, Rommie E.
  Journal of Chemical Theory and Computation (2014), 10 (11), 5047-5056CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
  Anal. of macromol./small-mol. binding pockets can provide important insights into mol. recognition and receptor dynamics. Since its release in 2011, the POVME (POcket Vol. MEasurer) algorithm has been widely adopted as a simple-to-use tool for measuring and characterizing pocket vols. and shapes. The authors here present POVME 2.0, which is an order of magnitude faster, has improved accuracy, includes a graphical user interface, and can produce volumetric d. maps for improved pocket anal. To demonstrate the utility of the algorithm, the authors use it to analyze the binding pocket of RNA editing ligase 1 from the unicellular parasite Trypanosoma brucei, the etiol. agent of African sleeping sickness. The POVME anal. characterizes the full dynamics of a potentially druggable transient binding pocket and so may guide future antitrypanosomal drug-discovery efforts. The authors are hopeful that this new version will be a useful tool for the computational- and medicinal-chemist community.
22. 22
  Clémençon, B.; Lüscher, B. P.; Hediger, M. A. Establishment of a Novel Microscale Thermophoresis Ligand-Binding Assay for Characterization of SLC Solute Carriers Using Oligopeptide Transporter PepT1 (SLC15 Family) as a Model System. J. Pharmacol. Toxicol. Methods 2018, 92, 67, DOI: 10.1016/j.vascn.2018.03.004
  22
  Establishment of a novel microscale thermophoresis ligand-binding assay for characterization of SLC solute carriers using oligopeptide transporter PepT1 (SLC15 family) as a model system
  Clemencon, Benjamin; Luscher, Benjamin P.; Hediger, Matthias A.
  Journal of Pharmacological and Toxicological Methods (2018), 92 (), 67-76CODEN: JPTMEZ; ISSN:1056-8719. (Elsevier)
  Membrane proteins represent roughly one third of the human proteome and many of them serve as targets of therapeutic drugs. An exception is the SLC solute carrier superfamily with only a handful of approved drugs targeting SLCs. Indeed, for many of the SLCs, the natural transport substrates are still unknown. A major limitation for SLCs has been the difficulty to thoroughly characterize these multimembrane spanning proteins. The intrinsic properties of membrane proteins with alternative hydrophobic and hydrophilic domains lead to instability, making the purifn. tasks even more challenging compared to sol. proteins. This issue also holds true for conventional ligand-binding assays (LBAs) which usually require high-quality, pure and concd. protein samples. Herein, we report a novel binding assay strategy to overcome these issues, taking advantage of a unique combination of yeast expression and microscale thermophoresis (MST). Following yeast overexpression of SLC15A1/PepT1 ortholog from moss Physcomitrella patens, PepTPp, which exhibits remarkable similarity to human PepT1, the approach was validated using dipeptide glycylsarcosine (Gly-Sar) and antiviral prodrug valacyclovir as test substrates. The originality of our approach is based on the comparative anal. of solubilized total membrane prepns. with or without expression of the SLC target of interest, using a yeast strain (S. cerevisiae), in which the corresponding endogenous SLC homolog is depleted. MST is a recently developed technique that takes advantage of the properties of biomols. in soln. to migrate along a temp. gradient. Importantly, this migration is affected by substrate binding. It is being monitored by fluorescence using labeled SLC mols. in the presence of different ligand concns. We herein report a novel MST/yeast-based method to characterize binding of ligands to SLCs without the need for a prior SLC-purifn. step. For validation purposes, we used a close eukaryotic homolog of the human H+-coupled oligopeptide transporter PepT1 (SLC15A1) that mediates uptake of di-tripeptides and peptide-like drugs as a test model. This approach allowed the successful confirmation of the binding of Gly-Sar at the mM range and revealed for the first time the KD of the antiviral prodrug valacyclovir to the PepT1 homolog at around 50 μM. This novel LBA approach is independent of protein purifn. It is suitable for drug discovery as it is upscalable to high throughput compd. screening. It works well for SLC transporters which are underrepresented targets due to their difficulties to study them. Moreover, this approach could make a significant contribution toward "deorphanization" of SLCs, revealing their transport substrates.
23. 23
  Malle, E.; Zhou, H.; Neuhold, J.; Spitzenberger, B.; Klepsch, F.; Pollak, T.; Bergner, O.; Ecker, G. F.; Stolt-Bergner, P. C. Random Mutagenesis of the Prokaryotic Peptide Transporter YdgR Identifies Potential Periplasmic Gating Residues. J. Biol. Chem. 2011, 286, 23121, DOI: 10.1074/jbc.M111.239657
  There is no corresponding record for this reference.
24. 24
  Kulkarni, A. A.; Haworth, I. S.; Lee, V. H. L. Transmembrane Segment 5 of the Dipeptide Transporter hPepT1 Forms a Part of the Substrate Translocation Pathway. Biochem. Biophys. Res. Commun. 2003, 306, 177, DOI: 10.1016/S0006-291X(03)00926-4
  There is no corresponding record for this reference.
25. 25
  Kulkarni, A. A.; Haworth, I. S.; Uchiyama, T.; Lee, V. H. L. Analysis of Transmembrane Segment 7 of the Dipeptide Transporter hPepT1 by Cysteine-scanning Mutagenesis. J. Biol. Chem. 2003, 278, 51833, DOI: 10.1074/jbc.M308356200
  There is no corresponding record for this reference.
26. 26
  Xu, L.; Haworth, I. S.; Kulkarni, A. A.; Bolger, M. B.; Davies, D. L. Mutagenesis and Cysteine Scanning of Transmembrane Domain 10 of the Human Dipeptide Transporter. Pharm. Res. 2009, 26, 2358, DOI: 10.1007/s11095-009-9952-9
  There is no corresponding record for this reference.
27. 27
  Woestenenk, E. A.; Hammarström, M.; van den Berg, S.; Härd, T.; Berglund, H. His tag Effect on Solubility of Human Proteins Produced in Escherichia coli: a Comparison between Four Expression Vectors. J. Struct. Funct. Genomics 2004, 5, 217, DOI: 10.1023/B:jsfg.0000031965.37625.0e
  27
  His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors
  Woestenenk, Esmeralda A.; Hammarstroem, Martin; van den Berg, Susanne; Haerd, Torleif; Berglund, Helena
  Journal of Structural and Functional Genomics (2004), 5 (3), 217-229CODEN: JSFGAW; ISSN:1345-711X. (Kluwer Academic Publishers)
  We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at total recombinant protein prodn. levels per g dry cell wt., soly. of the target proteins, and yield of sol. and total protein when purified by immobilized metal ion affinity purifn. It was found that, in general, both N- and C-terminal His6 tags have a noticeable neg. effect on protein soly., but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this neg. effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiol. conditions. The highest protein prodn. levels and yield of purified protein were obtained from a construct with a C-terminal His tag. We also observe a large variation in cell growth rate, which we detd. to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the prodn. level, soly. and tertiary structure content of the target proteins.
28. 28
  Flayhan, A.; Mertens, H. D. T.; Ural-Blimke, Y.; Martinez Molledo, M.; Svergun, D. I.; Löw, C. Saposin Lipid Nanoparticles: A Highly Versatile and Modular Tool for Membrane Protein Research. Structure 2018, 26, 345, DOI: 10.1016/j.str.2018.01.007
  28
  Saposin Lipid Nanoparticles: A Highly Versatile and Modular Tool for Membrane Protein Research
  Flayhan, Ali; Mertens, Haydyn D. T.; Ural-Blimke, Yonca; Martinez Molledo, Maria; Svergun, Dmitri I.; Loew, Christian
  Structure (Oxford, United Kingdom) (2018), 26 (2), 345-355.e5CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
  Saposin-derived lipid nanoparticles (SapNPs) are a new alternative tool for membrane protein reconstitution. Here we demonstrate the potential and advantages of SapNPs. We show that SapA has the lowest lipid specificity for SapNP formation. These nanoparticles are modular and offer a tunable range of size and compn. depending on the stoichiometric ratio of lipid and saposin components. They are stable and exhibit features typical of lipid-bilayer systems. Our data suggest that SapNPs are versatile and can adapt to membrane proteins of various sizes and architectures. Using SapA and various types of lipids we could reconstitute membrane proteins of different transmembrane cross-sectional areas (from 14 to 56 transmembrane α helixes). SapNP-reconstituted proteins bound their resp. ligands and were more heat stable compared with the detergent-solubilized form. Moreover, SapNPs encircle membrane proteins in a compact way, allowing structural investigations of small membrane proteins in a detergent-free environment using small-angle X-ray scattering.
29. 29
  Pardon, E.; Laeremans, T.; Triest, S.; Rasmussen, S.; Wohlkönig, A.; Ruf, A.; Muyldermans, S.; Hol, W. G. J.; Kobilka, B. K.; Steyaert, J. A General Protocol for the Generation of Nanobodies for Structural Biology. Nat. Protoc. 2014, 9, 674, DOI: 10.1038/nprot.2014.039
  29
  A general protocol for the generation of Nanobodies for structural biology
  Pardon, Els; Laeremans, Toon; Triest, Sarah; Rasmussen, Soren G. F.; Wohlkonig, Alexandre; Ruf, Armin; Muyldermans, Serge; Hol, Wim G. J.; Kobilka, Brian K.; Steyaert, Jan
  Nature Protocols (2014), 9 (3), 674-693CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
  There is growing interest in using antibodies as auxiliary tools to crystallize proteins. Here we describe a general protocol for the generation of Nanobodies to be used as crystn. chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins and complexes thereof. Our technol. has a competitive advantage over other recombinant crystn. chaperones in that we fully exploit the natural humoral response against native antigens. Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage display of in vivo-matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples illustrate that the outlined procedures are robust, making it possible to solve by Nanobody-assisted X-ray crystallog. in a time span of 6-12 mo.
30. 30
  Farrell, I. S.; Toroney, R.; Hazen, J. L.; Mehl, R. A.; Chin, J. W. Photo-Cross-Linking Interacting Proteins with a Genetically Encoded Benzophenone. Nat. Methods 2005, 2, 377, DOI: 10.1038/nmeth0505-377
  30
  Photo-cross-linking interacting proteins with a genetically encoded benzophenone
  Farrell, Ian S.; Toroney, Rebecca; Hazen, Jennifer L.; Mehl, Ryan A.; Chin, Jason W.
  Nature Methods (2005), 2 (5), 377-384CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
  A major challenge in understanding the networks of interactions that control cell and organism function is the definition of protein interactions. Solid-phase peptide synthesis has allowed the photo-crosslinkable amino acid p-benzoyl-L-phenylalanine to be site-specifically incorporated into peptide chains, to facilitate the definition of peptide-ligand complexes. The method, however, is limited to the in vitro study of peptides and small proteins. An innovative develpoment allows the incorporation of a site-specific photo-cross-linker into virtually any protein that can be expressed in Escherichia coli, thereby promoting in vivo or in vitro crosslinking of proteins. The method relies on an orthogonal aminoacyl tRNA synthetase-tRnACUA pair that incorporates pBpa at the position encoded by the amber codon (UAG) in any gene transformed into E. coli. The system described in this protocol uses two plasmids: a p15A-based plasmid to express the orthogonal tRNA and synthetase pair (pDULE) and a second plasmid contg. an amber mutant of the gene of interest. To produce the photo-cross-linker-contg. protein, cultures of E. coli carrying both plasmids are grown in the presence of the unnatural amino acid. To photo-cross-link the protein to its binding partner in vivo or in vitro, cells or purified proteins, resp., are exposed to UV light.
31. 31
  Bowler, M. W.; Nurizzo, D.; Barrett, R.; Beteva, A.; Bodin, M.; Caserotto, H.; Delagenière, S.; Dobias, F.; Flot, D.; Giraud, T.; Guichard, N.; Guijarro, M.; Lentini, M.; Leonard, G. A.; McSweeney, S.; Oskarsson, M.; Schmidt, W.; Snigirev, A.; von Stetten, D.; Surr, J.; Svensson, O.; Theveneau, P.; Mueller-Dieckmann, C. MASSIF-1: a Beamline Dedicated to the Fully Automatic Characterization and Data Collection from Crystals of Biological Macromolecules. J. Synchrotron Radiat. 2015, 22, 1540, DOI: 10.1107/S1600577515016604
  31
  MASSIF-1: a beamline dedicated to the fully automatic characterization and data collection from crystals of biological macromolecules
  Bowler Matthew W; Nurizzo Didier; Barrett Ray; Beteva Antonia; Bodin Marjolaine; Caserotto Hugo; Delageniere Solange; Dobias Fabian; Flot David; Giraud Thierry; Guichard Nicolas; Guijarro Mattias; Lentini Mario; Leonard Gordon A; McSweeney Sean; Oskarsson Marcus; Schmidt Werner; Snigirev Anatoli; von Stetten David; Surr John; Svensson Olof; Theveneau Pascal; Mueller-Dieckmann Christoph
  Journal of synchrotron radiation (2015), 22 (6), 1540-7 ISSN:.
  MASSIF-1 (ID30A-1) is an ESRF undulator beamline operating at a fixed wavelength of 0.969 ÅA (12.8 keV) that is dedicated to the completely automatic characterization of and data collection from crystals of biological macromolecules. The first of the ESRF Upgrade MASSIF beamlines to be commissioned, it has been open since September 2014, providing a unique automated data collection service to academic and industrial users. Here, the beamline characteristics and details of the new service are outlined.
32. 32
  Kabsch, W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125, DOI: 10.1107/S0907444909047337
  32
  Software XDS for image rotation, recognition and crystal symmetry assignment
  Kabsch, Wolfgang
  Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 125-132CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
  The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic detn. of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the no. of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.
33. 33
  McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl. Crystallogr. 2007, 40, 658, DOI: 10.1107/S0021889807021206
  33
  Phaser crystallographic software
  McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.; Winn, Martyn D.; Storoni, Laurent C.; Read, Randy J.
  Journal of Applied Crystallography (2007), 40 (4), 658-674CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)
  Phaser is a program for phasing macromol. crystal structures by both mol. replacement and exptl. phasing methods. The novel phasing algorithms implemented in Phaser have been developed using max. likelihood and multivariate statistics. For mol. replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solns. from noise, and for single-wavelength anomalous dispersion exptl. phasing, the new algorithms, which account for correlations between F+ and F-, give better phases (lower mean phase error with respect to the phases given by the refined structure) than those that use mean F and anomalous differences ΔF. One of the design concepts of Phaser was that it be capable of a high degree of automation. To this end, Phaser (written in C++) can be called directly from Python, although it can also be called using traditional CCP4 keyword-style input. Phaser is a platform for future development of improved phasing methods and their release, including source code, to the crystallog. community.
34. 34
  Adams, P. D.; Afonine, P. V.; Bunkóczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L.-W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. PHENIX: a Comprehensive Python-Based System for Macromolecular Structure Solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213, DOI: 10.1107/S0907444909052925
  34
  PHENIX: a comprehensive Python-based system for macromolecular structure solution
  Adams, Paul D.; Afonine, Pavel V.; Bunkoczi, Gabor; Chen, Vincent B.; Davis, Ian W.; Echols, Nathaniel; Headd, Jeffrey J.; Hung, Li Wei; Kapral, Gary J.; Grosse-Kunstleve, Ralf W.; McCoy, Airlie J.; Moriarty, Nigel W.; Oeffner, Robert; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Zwart, Peter H.
  Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 213-221CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
  A review. Macromol. X-ray crystallog. is routinely applied to understand biol. processes at a mol. level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromol. crystallog. structure soln. with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
35. 35
  Emsley, P.; Cowtan, K. Coot: Model-Building Tools for Molecular Graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126, DOI: 10.1107/S0907444904019158
  35
  Coot: model-building tools for molecular graphics
  Emsley, Paul; Cowtan, Kevin
  Acta Crystallographica, Section D: Biological Crystallography (2004), D60 (12, Pt. 1), 2126-2132CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
  CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for x-ray structure soln., structure comparison and anal., and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as 'Coot'.
36. 36
  Cianci, M.; Bourenkov, G.; Pompidor, G.; Karpics, I.; Kallio, J.; Bento, I.; Roessle, M.; Cipriani, F.; Fiedler, S.; Schneider, T. R. P13, the EMBL Macromolecular Crystallography Beamline at the Low-Emittance PETRA III Ring for High- and Low-Energy Phasing with Variable Beam Focusing. J. Synchrotron Radiat. 2017, 24, 323, DOI: 10.1107/S1600577516016465
  36
  P13, the EMBL macromolecular crystallography beamline at the low-emittance PETRA III ring for high- and low-energy phasing with variable beam focusing
  Cianci, Michele; Bourenkov, Gleb; Pompidor, Guillaume; Karpics, Ivars; Kallio, Johanna; Bento, Isabel; Roessle, Manfred; Cipriani, Florent; Fiedler, Stefan; Schneider, Thomas R.
  Journal of Synchrotron Radiation (2017), 24 (1), 323-332CODEN: JSYRES; ISSN:1600-5775. (International Union of Crystallography)
  The macromol. crystallog. P13 beamline is part of the European Mol. Biol. Lab. Integrated Facility for Structural Biol. at PETRA III (DESY, Hamburg, Germany) and has been in user operation since mid-2013. P13 is tunable across the energy range from 4 to 17.5 keV to support crystallog. data acquisition exploiting a wide range of elemental absorption edges for exptl. phase detn. An adaptive Kirkpatrick-Baez focusing system provides an X-ray beam with a high photon flux and tunable focus size to adapt to diverse exptl. situations. Data collections at energies as low as 4 keV (λ = 3.1 Å) are possible due to a beamline design minimizing background and maximizing photon flux particularly at low energy (up to 1011 photons s-1 at 4 keV), a custom calibration of the PILATUS 6M-F detector for use at low energies, and the availability of a helium path. At high energies, the high photon flux (5.4 × 1011 photons s-1 at 17.5 keV) combined with a large area detector mounted on a 2θ arm allows data collection to sub-at. resoln. (0.55 Å). A peak flux of about 8.0 × 1012 photons s-1 is reached at 11 keV. Automated sample mounting is available by means of the robotic sample changer 'MARVIN' with a dewar capacity of 160 samples. In close proximity to the beamline, labs. have been set up for sample prepn. and characterization; a lab. specifically equipped for on-site heavy atom derivatization with a library of more than 150 compds. is available to beamline users.
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  Pence, H. E.; Williams, A. ChemSpider: An Online Chemical Information Resource. J. Chem. Educ. 2010, 87, 1123, DOI: 10.1021/ed100697w
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  ChemSpider: An Online Chemical Information Resource
  Pence, Harry E.; Williams, Antony
  Journal of Chemical Education (2010), 87 (11), 1123-1124CODEN: JCEDA8; ISSN:0021-9584. (American Chemical Society and Division of Chemical Education, Inc.)
  ChemSpider is a free, online chem. database offering access to phys. and chem. properties, mol. structure, spectral data, synthetic methods, safety information, and nomenclature for almost 25 million unique chem. compds. sourced and linked to almost 400 sep. data sources on the Web. ChemSpider is quickly becoming the primary chem. Internet portal and it can be very useful for both chem. teaching and research.
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  Comparative protein modeling by satisfaction of spatial restraints
  Sali, Andrej; Blundell, Tom L.
  Journal of Molecular Biology (1993), 234 (3), 779-815CODEN: JMOBAK; ISSN:0022-2836.
  The authors describe a comparative protein modeling method designed to find the most probable structure for a sequence given its alignment with related structures. The three-dimensional (3D) model is obtained by optimally satisfying spatial restraints derived from the alignment and expressed as probability d. functions (pdfs) for the features restrained. For example, the probabilities for main-chain conformations of a modelled residue may be restrained by its residue type, main-chain conformation of an equiv. residue in a related protein, and the local similarity between the two sequences. Several such pdfs are obtained from the correlations between structural features in 17 families of homologous proteins which have been aligned on the basis of their 3D structures. The pdfs restrain Cα-Cα distances, main-chain N-O distances, main-chain and side-chain dihedral angles. A smoothing procedure is used in the derivation of these relationships to minimize the problem of a sparse database. The 3D model of a protein is obtained by optimization of the mol. pdf such that the model violates the input restraints as little as possible. The mol. pdf is derived as a combination of pdfs restraining individual spatial features of the whole mol. The optimization procedure is a variable target function method that applies the conjugate gradients algorithm to positions of all non-hydrogen atoms. The method is automated and is illustrated by the modeling of trypsin from two other serine proteinases.
44. 44
  Zimmermann, L.; Stephens, A.; Nam, S.-Z.; Rau, D.; Kübler, J.; Lozajic, M.; Gabler, F.; Söding, J.; Lupas, A. N.; Alva, V. A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. J. Mol. Biol. 2018, 430, 2237, DOI: 10.1016/j.jmb.2017.12.007
  44
  A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core
  Zimmermann, Lukas; Stephens, Andrew; Nam, Seung-Zin; Rau, David; Kuebler, Jonas; Lozajic, Marko; Gabler, Felix; Soeding, Johannes; Lupas, Andrei N.; Alva, Vikram
  Journal of Molecular Biology (2018), 430 (15), 2237-2243CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
  The MPI Bioinformatics Toolkit (https://toolkit.tuebingen.mpg.de) is a free, one-stop web service for protein bioinformatic anal. It currently offers 34 interconnected external and inhouse tools, whose functionality covers sequence similarity searching, alignment construction, detection of sequence features, structure prediction, and sequence classification. This breadth has made the Toolkit an important resource for exptl. biol. and for teaching bioinformatic inquiry. Recently, we replaced the first version of the Toolkit, which was released in 2005 and had served around 2.5 million queries, with an entirely new version, focusing on improved features for the comprehensive anal. of proteins, as well as on promoting teaching. For instance, our popular remote homol. detection server, HHpred, now allows pairwise comparison of two sequences or alignments and offers addnl. profile HMMs for several model organisms and domain databases. Here, we introduce the new version of our Toolkit and its application to the anal. of proteins.
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  45
  SWISS-MODEL and the Swiss-PdbViewer. An environment for comparative protein modeling
  Guex, Nicolas; Peitsch, Manuel C.
  Electrophoresis (1997), 18 (15), 2714-2723CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH)
  Comparative protein modeling is increasingly gaining interest since it is of great assistance during the rational design of mutagenesis expts. The availability of this method, and the resulting models, has however been restricted by the availability of expensive computer hardware and software. To overcome these limitations, the authors have developed an environment for comparative protein modeling that consists of SWISS-MODEL, a server for automated comparative protein modeling and of the SWISS-PdbViewer, a sequence to structure workbench. The Swiss-PdbViewer not only acts as a client for SWISS-MODEL, but also provides a large selection of structure anal. and display tools. In addn., the authors provide the SWISS-MODEL Repository, a database contg. more than 3500 automatically generated protein models. By making such tools freely available to the scientific community, the authors hope to increase the use of protein structures and models in the process of expt. design.
46. 46
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47. 47
  Beale, J. H.; Parker, J. L.; Samsudin, F.; Barrett, A. L.; Senan, A.; Bird, L. E.; Scott, D.; Owens, R. J.; Sansom, M. S. P.; Tucker, S. J.; Meredith, D.; Fowler, P. W.; Newstead, S. Crystal Structures of the Extracellular Domain from PepT1 and PepT2 Provide Novel Insights into Mammalian Peptide Transport. Structure 2015, 23, 1889, DOI: 10.1016/j.str.2015.07.016
  47
  Crystal Structures of the Extracellular Domain from PepT1 and PepT2 Provide Novel Insights into Mammalian Peptide Transport
  Beale, John H.; Parker, Joanne L.; Samsudin, Firdaus; Barrett, Anne L.; Senan, Anish; Bird, Louise E.; Scott, David; Owens, Raymond J.; Sansom, Mark S. P.; Tucker, Stephen J.; Meredith, David; Fowler, Philip W.; Newstead, Simon
  Structure (Oxford, United Kingdom) (2015), 23 (10), 1889-1899CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
  Mammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter contg. a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two Ig-like folds connected in tandem, providing structural insight into mammalian peptide transport. Functional and biophys. studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clustering proteolytic activity to the site of peptide transport in eukaryotic cells.
Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b11343.
- Nanobody screening for DtpA binding; electron density maps of DtpA; DtpA-N00 binding site; stereoimage of the ligand-binding site with valganciclovir; AK-AMCA uptake and concentration-dependent competition assay with DtpA; principal component analysis of physico-chemical data for di- and tripeptide ligands; binding curves derived from microscale thermophoresis experiment for DtpA and the DtpA-N00 complex; cross-linking of DtpA and N00 in a lipid environment; thermal stability and binding affinity analysis of DtpA mutants; sequence alignment of DtpA and hPepT1; ligand-binding site of DtpA and hPepT1 with valganciclovir; key residues for function in TM5, TM7, and TM10 highlighted in the hPepT1 homology model; thermal stability (T_m), binding affinity (K_d), and AK-AMCA uptake results for DtpA and DtpA-N00; binding affinity results for DtpA mutants; references (PDF)
- ja8b11343_si_001.pdf (2.02 MB)
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Structure of Prototypic Peptide Transporter DtpA from E. coli in Complex with Valganciclovir Provides Insights into Drug Binding of Human PepT1Click to copy article linkArticle link copied!

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Abstract

Introduction

Results and Discussion

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Concluding Remarks

Methods

Chemicals

Gene Construction, Protein Expression, and Purification of DtpA

Selection, Expression, and Purification of Nanobodies Against DtpA

Analytical Gel Filtration Assay

Evaluation of Conformational or Linear Epitope Binding

Cross-Linking of DtpA and N00 in the Lipid Bilayer

Crystallization and Structure Determination

Analysis of the Binding Site Volume

Thermal Stability Assay

Binding Affinity Assay

In Vivo Uptake and Competition Assay

Principal Component Analysis

Model Building of Human PepT1 (hPepT1, SLC15A1) with Valganciclovir

Supporting Information

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Acknowledgments

References

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Supporting Information

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Structure of Prototypic Peptide Transporter DtpA from E. coli in Complex with Valganciclovir Provides Insights into Drug Binding of Human PepT1
Click to copy article linkArticle link copied!