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Supramolecular Interactions of Teixobactin Analogues in the Crystal State
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The Journal of Organic Chemistry

Cite this: J. Org. Chem. 2024, 89, 7, 5104–5108
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https://doi.org/10.1021/acs.joc.3c02617
Published March 20, 2024

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Abstract

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This Note presents the X-ray crystallographic structure of the N-methylated teixobactin analogue N-Me-d-Gln4,Lys10-teixobactin (1). Eight peptide molecules comprise the asymmetric unit, with each peptide molecule binding a chloride anion through hydrogen bonding with the amide NH group of residues 7, 8, 10, and 11. The peptide molecules form hydrogen-bonded antiparallel β-sheet dimers in the crystal lattice, with residues 1–3 comprising the dimerization interface. The dimers further assemble end-to-end in the crystal lattice.

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Teixobactin is a peptide antibiotic that exhibits remarkable efficacy against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, drug-resistant Streptococcus pneumonia, and vancomycin-resistant Enterococci. (1) Teixobactin’s mode of action involves binding to lipid II and related peptidoglycan precursors and disrupting the bacterial cell membrane. Teixobactin is an undecapeptide consisting of an N-terminal linear tail encompassing residues 1–7 and a C-terminal macrocyclic ring spanning residues 8–11 (Figure 1). The linear tail consists of N-Me-d-Phe1, Ile2, Ser3, d-Gln4, d-allo-Ile5, Ile6, and Ser7, and the macrocyclic ring consists of d-Thr8, Ala9, the cyclic arginine analogue allo-End10, and Ile11. The C-terminus of Ile11 and the hydroxy group of d-Thr8 form an ester bond, creating a 13-membered lactone ring.

Figure 1

Figure 1. Chemical structures of teixobactin, N-methylated d-Gln4 analogues of teixobactin, and a truncated teixobactin analogue.

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Supramolecular assembly is central to the mechanism of action of teixobactin. (2,3) Two teixobactin molecules come together to form an antiparallel β-sheet dimer, which binds the pyrophosphate group of lipid II. The N-terminal tails comprise the dimerization interface and adopt a conformation in which the residues N-Me-d-Phe1, Ile2, d-allo-Ile5, and Ile6 form a hydrophobic surface that interacts with the bacterial cell membrane. The allo-End10 residue, although not essential for binding and antibiotic activity, makes additional contacts with the pyrophosphate and MurNAc groups of lipid II that impart enhanced affinity and antibiotic activity. Further supramolecular assembly leads to the clustering of lipid II and the lysis of Gram-positive bacteria.
Our laboratory has pioneered the use of X-ray crystallography to gain insights into the structure and supramolecular interactions of teixobactin. Although teixobactin itself is highly prone to amyloid-like aggregation and cannot be crystallized, our laboratory has found that N-methylation at d-Gln4 blocks the aggregation and permits crystallization. The resulting N-methylated teixobactin analogues exhibit little or no antibiotic activity. We previously observed that teixobactin analogue 2 undergoes supramolecular assembly to form antiparallel β-sheet dimers that bind sulfate anions and further form fibril-like assemblies (PDB 6E00). (4) We also observed antiparallel β-sheet dimers in the X-ray crystallographic structure of teixobactin analogue 3, and we observed that these dimers bind chloride anions (PDB 6PSL). (5) We further observed chloride anion binding in the X-ray crystallographic structure of truncated teixobactin analogue 4 (CCDC 1523518). (6) In the current study, we report the X-ray crystallographic structure of teixobactin analogue 1 and describe its supramolecular interactions in the crystal state.
The X-ray crystallographic structure of teixobactin analogue 1 reveals eight peptide molecules in the asymmetric unit, with each peptide molecule binding a chloride anion (Figure 2). The eight peptide molecules adopt similar conformations, with a backbone RMSD of 0.58 Å. Each peptide molecule forms an amphiphilic surface in which the side chains of N-Me-d-Phe1, Ile2, d-allo-Ile5, Ile6, and Ile11 and the β-methyl of d-Thr8 create a hydrophobic surface and the side chains of residues Ser3, N-Me-d-Gln4, Ser7, and Lys10 create a hydrophilic surface. The carbonyl groups of the macrocycle align, and the amide NH groups of Ser7, d-Thr8, Lys10, and Ile11 hydrogen bond to the chloride anion. The oxygen atom comprising the ester linkage also faces toward the chloride ion. The amide NH group of Ala9 hydrogen bonds to the hydroxy group of Ser7.

Figure 2

Figure 2. X-ray crystallographic structure of teixobactin analogue 1 (PDB 8U78). (A) Overlay of the eight peptide molecules binding chloride anions in the asymmetric unit. (B) Side view and (C) end view of a representative peptide molecule.

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The peptide molecules form hydrogen-bonded antiparallel β-sheet dimers in the crystal lattice (Figure 3). In each dimer, residues 1–3 come together through hydrogen bonding interactions to create the antiparallel β-sheet structure. The N-methyl groups of N-Me-d-Gln4 point outward from the hydrogen-bonding interface. The dimer is amphiphilic, with the top surface shown in Figure 3 being hydrophilic and the bottom surface being hydrophobic.

Figure 3

Figure 3. Hydrogen-bonded antiparallel β-sheet dimer in the crystal lattice of teixobactin analogue 1.

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The dimers further assemble end-to-end in the crystal lattice, as illustrated in Figure 4. In this arrangement, the macrocycle of each molecule is in contact with the N-terminus of another molecule in the adjacent dimer, with the carbonyl group of Ala9 hydrogen bonding to the N-methylammonium group. Two macrocycles are in proximity at each interface between dimers, with the bound chloride anions being ca. 7 Å apart. The end-to-end assembly of dimers is amphiphilic, and the hydrophobic surfaces pack together in the crystal lattice.

Figure 4

Figure 4. End-to-end dimer assemblies and their packing in the crystal lattice. (A) Top view. (B) Side view. (C) Hydrophobic packing in the crystal lattice.

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The dimers and other supramolecular interactions of N-Me-d-Gln4,Lys10-teixobactin (1) observed in the crystal state differ in several ways from those of N-methylated teixobactin analogues 2 and 3 (Figure 5). In the antiparallel β-sheet dimer of teixobactin analogue 1, residues 1–3 hydrogen bond, with N-Me-d-Phe1 pairing with Ser3. Analogue 2 also forms an antiparallel β-sheet dimer, but there is more overlap of the strands with residues 1–6 forming the dimer interface (PDB 6E00). The dimers further assemble through antiparallel β-sheet interactions involving residues 3–7 of their outer edges. Analogue 3 forms an antiparallel β-sheet dimer with residues 3–5 forming the dimer interface (PDB 6PSL). In the dimers formed by analogues 2 and 3, the N-terminus of one peptide molecule helps bind the anion that is grasped by the C-terminal region of the other molecule. In the dimers formed by analogue 1, however, only the C-terminal region participates in anion binding. The dimers of analogues 1 and 2 further assemble in the lattice to create amphiphilic sheets that pack through hydrophobic interactions. The dimers of analogue 3 further assemble through hydrophobic interactions to create threefold symmetrical rods that extend through the crystal lattice.

Figure 5

Figure 5. Chemical drawings of X-ray crystallographic structures of teixobactin analogues and their antiparallel β-sheet dimer interactions. (A) Analogue 1. (B) Analogue 2. (C) Analogue 3. (D) Analogue 4.

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Despite these differences, similarities exist across all of the X-ray crystallographic structures that we have determined. The analogues 1–3 each come together through antiparallel β-sheet interactions to create amphiphilic β-sheet dimers. The dimerization of these analogues likely reflects the propensity of teixobactin to form dimers and higher-order assemblies that interact with bacterial cell membranes through hydrophobic interactions. Analogues 1–3 and truncated analogue 4 (CCDC 1523518) each bind anions in the crystal state, with analogues 1, 3, and 4 binding chloride and analogue 2 binding sulfate. The binding of these anions likely reflects the propensity of teixobactin to bind the pyrophosphate groups of lipid II and related cell wall precursors.
Supramolecular assembly is integral to the mechanism of action of teixobactin. N-Methylation at d-Gln4 helps prevent uncontrolled aggregation, permits the crystallization of teixobactin analogues, and allows the observation of conformation and supramolecular interactions at atomic resolution. In the crystal state, the teixobactin analogues adopt an amphiphilic conformation and form antiparallel β-sheet dimers though hydrogen bonding between the N-terminal tails. The macrocycles formed by residues 8–11 bind anions through hydrogen bonding. The hydrophobic surfaces of the dimers further pack through hydrophobic interactions. These observations further support a model in which teixobactin binds to Gram-positive bacteria through interactions of its hydrophobic side chains with the bacterial cell membrane, undergoes supramolecular assembly through β-sheet interactions, and binds the pyrophosphate groups of lipid II and related cell-wall precursors.

Experimental Section

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General Information

Methylene chloride (CH2Cl2) was passed through alumina under argon prior to use. Amine-free N,N-dimethylformamide (DMF) was purchased from Alfa Aesar. Fmoc-d-allo-Ile-OH was purchased from Santa Cruz Biotechnology. Fmoc-N-Me-d-Gln(Trt)-OH was purchased from ChemPep. Other protected amino acids were purchased from CHEM-IMPEX. Preparative reversed-phase HPLC was performed on a Rainin Dynamax instrument equipped with an Agilent Zorbax SB-C18 column. Analytical reverse-phase HPLC was performed on an Agilent 1260 Infinity II instrument equipped with a Phenomonex Aeris PEPTIDE 2.6μ XB-C18 column. HPLC grade acetonitrile (MeCN) and deionized water (18 MΩ) containing 0.1% trifluoroacetic acid (TFA) were used as solvents for both preparative and analytical reverse-phase HPLC. Deionized water (18 MΩ) was obtained from a Barnstead NANOpure Diamond purification system or a ThermoScientific Barnstead GenPure Pro water purification system. Glass solid-phase peptide synthesis vessels with fritted disks and BioRad Polyprep columns were used for the solid-phase peptide synthesis. Teixobactin analogue 1 was prepared and studied as the trifluoroacetate salt.

Synthesis and Crystallization of N-Me-d-Gln4,Lys10-teixobactin (1)

We synthesized N-Me-d-Gln4,Lys10-teixobactin (1) as the trifluoroacetate (TFA) salt as described. (7) Crystallization was performed by using standard protein crystallography techniques. Briefly, N-Me-d-Gln4,Lys10-teixobactin (1) was dissolved in 18 MΩ deionized H2O at a concentration of 20 mg/mL. Suitable crystallization conditions were identified by screening experiments in a 96-well plate format with the aid of using screening kits from Hampton Research (including PEG/Ion, Index, and Crystal Screen kits). Each well was loaded with 100 μL of a solution from the kits. Hanging drops were set up using a TTP Labtech Mosquito liquid handling instrument, with three 150 nL drops per well with 1:1, 1:2, and 2:1 ratios of well solution and peptide stock solution. Crystals grew in conditions containing chloride with PEG3350 as a precipitant.
To optimize the crystal growth, we tested various conditions that included PEG3350 and MgCl2. In this optimization process, we varied the concentrations of PEG3350 (from 15% to 30%) and MgCl2 (from 0.20 to 0.38 M) across a 4 × 6 matrix within a Hampton VDX 24-well plate. Hanging drops for the optimization experiments were prepared on glass slides by combining 1 or 2 μL of the N-Me-d-Gln4,Lys10-teixobactin solution with 1 or 2 μL of the well solution using ratios of 1:1, 2:1, and 1:2. Crystals were assessed for X-ray diffraction using a Rigaku Micromax-007 HF diffractometer equipped with a Cu anode.

X-ray Data Collection and Processing

Data collection was conducted using the BOS/B3 software at the Advanced Light Source (ALS) on beamline 8.2.2. We collected X-ray diffraction at the longest wavelength possible (2.0663 Å, 6000 eV) to allow for the use of chloride K-edge X-ray absorption (2822 eV) for single anomalous diffraction (SAD) phasing. Three data sets were acquired from three different crystals. Two sets of 360 images were acquired per crystal with 1.0° rotation intervals (equivalent to two complete 360° rotations). The data sets were processed using XDS, (8) and the resulting data sets were merged employing BLEND. (9) To determine the structure, we used SAD phasing, implementing the Hybrid Substructure Search (HySS) (10) module from the Phenix suite. (11) The anomalous signals from the chloride ions were used in this process. Initial electron density maps were generated by incorporating the chloride coordinates as starting positions in Autosol. (12) We also collected a higher-resolution data set at 0.9997 Å wavelength to allow the use of the molecular replacement with the structure determined from the 2.0663 Å data. X-ray diffraction data collection and processing information are summarized in Table S1.

X-ray Structure Solution and Refinement

The structure was refined using Phenix.refine, (13) and Coot (14) was used for model building. During refinement, all B-factors were refined isotropically and coordinates for hydrogen atoms were generated geometrically. For the nonproteinogenic amino acids (N-Me-d-Gln4, d-Thr8, and d-allo-Ile5), bond lengths, angles, and torsion restraints were generated using eLBOW (15) in the Phenix software. The resulting structure was successfully used for molecular replacement with the 0.9997 Å resolution data set, which was refined in a similar fashion. Pentaethylene glycol dimethyl ether was included in the refinement to fill the electron density associated with PEG3350. Final refinement and validation statistics are shown in Table S2 (PDB 8U78).

HPLC Conditions and MS Results

Analytical RP-HPLC was performed on a C18 column with an elution gradient of 5–67% CH3CN + 0.1% TFA over 15 min.

N-Me-d-Gln4,Lys10-teixobactin (1)

MS (ESI) m/z: [M + H]+ calcd for C59H100N13O15 1230.75, found 1230.5.

Data Availability

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The data underlying this study are available in the published article and its Supporting Information.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.joc.3c02617.

  • HPLC and MS characterization data and X-ray crystallographic statistics for N-Me-d-Gln4,Lys10-teixobactin (1) (PDF)

Accession Codes

Crystallographic coordinates of N-Me-d-Gln4,Lys10-teixobactin (1) were deposited into the Protein Data Bank (PDB) with code 8U78.

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
    • James S. Nowick - Department of Chemistry, University of California Irvine, Irvine, California 92697, United States;  Department of Pharmaceutical Sciences, University of California Irvine, Irvine, California 92697, United States;  Orcidhttps://orcid.org/0000-0002-2273-1029;  Email: [email protected]
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  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was supported by the National Institutes of Health (Grants AI121548 and AI137258). H.Y. acknowledges Allergan for fellowship support. We thank the Berkeley Center for Structural Biology (BCSB) of the Advanced Light Source (ALS) for synchrotron data collection. The BCSB is supported in part by the Howard Hughes Medical Institute. The ALS is supported by DOE Office of Science User Facility under contract no. DE-AC02-05CH11231. Previous versions of this manuscript are available in bioRxiv at https://doi.org/10.1101/2023.10.30.564786.

References

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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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    Foadi, J.; Aller, P.; Alguel, Y.; Cameron, A.; Axford, D.; Owen, R. L.; Armour, W.; Waterman, D. G.; Iwata, S.; Evans, G. Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2013, 69, 1617– 1632,  DOI: 10.1107/S0907444913012274
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    The availability of intense microbeam macromol. crystallog. beamlines at third-generation synchrotron sources has enabled data collection and structure soln. from microcrystals of <10 μm in size. The increased likelihood of severe radiation damage where microcrystals or particularly sensitive crystals are used forces crystallographers to acquire large nos. of data sets from many crystals of the same protein structure. The assocd. anal. and merging of multi-crystal data is currently a manual and time-consuming step. Here, a computer program, BLEND, that has been written to assist with and automate many of the steps in this process is described. It is demonstrated how BLEND has successfully been used in the soln. of a novel membrane protein.
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    This paper accompanies a lecture given at the 2003 CCP4 Study Weekend on exptl. phasing. The first part is an overview of the fundamentals of Patterson methods and direct methods with the audience of the CCP4 Study Weekend in mind. In the second part, a new hybrid substructure search is outlined.
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    Adams, P. D.; Afonine, P. V.; Bunkoczi, 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– 221,  DOI: 10.1107/S0907444909052925
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    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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    Terwilliger, T. C.; Adams, P. D.; Read, R. J.; McCoy, A. J.; Moriarty, N. W.; Grosse-Kunstleve, R. W.; Afonine, P. V.; Zwart, P. H.; Hung, L. W. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65, 582– 601,  DOI: 10.1107/S0907444909012098
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    Acta Crystallographica, Section D: Biological Crystallography (2009), 65 (6), 582-601CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    Ests. of the quality of exptl. maps are important in many stages of structure detn. of macromols. Map quality is defined here as the correlation between a map and the corresponding map obtained using phases from the final refined model. Here, ten different measures of exptl. map quality were examd. using a set of 1359 maps calcd. by re-anal. of 246 solved MAD, SAD and MIR data sets. A simple Bayesian approach to estn. of map quality from one or more measures is presented. It was found that a Bayesian estimator based on the skewness of the d. values in an electron-d. map is the most accurate of the ten individual Bayesian estimators of map quality examd., with a correlation between estd. and actual map quality of 0.90. A combination of the skewness of electron d. with the local correlation of r.m.s. d. gives a further improvement in estg. map quality, with an overall correlation coeff. of 0.92. The PHENIX AutoSol wizard carries out automated structure soln. based on any combination of SAD, MAD, SIR or MIR data sets. The wizard is based on tools from the PHENIX package and uses the Bayesian ests. of map quality described here to choose the highest quality solns. after exptl. phasing.
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    Afonine, P. V.; Grosse-Kunstleve, R. W.; Echols, N.; Headd, J. J.; Moriarty, N. W.; Mustyakimov, M.; Terwilliger, T. C.; Urzhumtsev, A.; Zwart, P. H.; Adams, P. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2012, 68, 352– 367,  DOI: 10.1107/S0907444912001308
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    Towards automated crystallographic structure refinement with phenix.refine
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    Acta Crystallographica, Section D: Biological Crystallography (2012), 68 (4), 352-367CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    Phenix.refine is a program within the PHENIX package that supports crystallog. structure refinement against exptl. data with a wide range of upper resoln. limits using a large repertoire of model parameterizations. It has several automation features and is also highly flexible. Several hundred parameters enable extensive customizations for complex use cases. Multiple user-defined refinement strategies can be applied to specific parts of the model in a single refinement run. An intuitive graphical user interface is available to guide novice users and to assist advanced users in managing refinement projects. X-ray or neutron diffraction data can be used sep. or jointly in refinement. phenix.refine is tightly integrated into the PHENIX suite, where it serves as a crit. component in automated model building, final structure refinement, structure validation and deposition to the wwPDB. This paper presents an overview of the major phenix.refine features, with extensive literature refs. for readers interested in more detailed discussions of the methods.
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    Features and development of Coot
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    Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
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    Moriarty, N. W.; Grosse-Kunstleve, R. W.; Adams, P. D. electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65, 1074– 1080,  DOI: 10.1107/S0907444909029436
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    electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation
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    The electronic Ligand Builder and Optimization Workbench (eLBOW) is a program module of the PHENIX suite of computational crystallog. software. It is designed to be a flexible procedure that uses simple and fast quantum-chem. techniques to provide chem. accurate information for novel and known ligands alike. A variety of input formats and options allow the attainment of a no. of diverse goals including geometry optimization and generation of restraints.

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  1. Jackson E. H. Brunicardi, James H. Griffin, Michael J. Ferracane, Adam G. Kreutzer, Jeramiah Small, Ana-Teresa Mendoza, Joseph W. Ziller, James S. Nowick. Structure–Activity Relationship Studies of the Peptide Antibiotic Clovibactin. The Journal of Organic Chemistry 2024, 89 (17) , 12479-12484. https://doi.org/10.1021/acs.joc.4c01414
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  • Abstract

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    Figure 1

    Figure 1. Chemical structures of teixobactin, N-methylated d-Gln4 analogues of teixobactin, and a truncated teixobactin analogue.

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    Figure 2

    Figure 2. X-ray crystallographic structure of teixobactin analogue 1 (PDB 8U78). (A) Overlay of the eight peptide molecules binding chloride anions in the asymmetric unit. (B) Side view and (C) end view of a representative peptide molecule.

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    Figure 3

    Figure 3. Hydrogen-bonded antiparallel β-sheet dimer in the crystal lattice of teixobactin analogue 1.

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    Figure 4

    Figure 4. End-to-end dimer assemblies and their packing in the crystal lattice. (A) Top view. (B) Side view. (C) Hydrophobic packing in the crystal lattice.

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    Figure 5

    Figure 5. Chemical drawings of X-ray crystallographic structures of teixobactin analogues and their antiparallel β-sheet dimer interactions. (A) Analogue 1. (B) Analogue 2. (C) Analogue 3. (D) Analogue 4.

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  • References

    This article references 15 other publications.

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      A new antibiotic kills pathogens without detectable resistance
      Ling, Losee L.; Schneider, Tanja; Peoples, Aaron J.; Spoering, Amy L.; Engels, Ina; Conlon, Brian P.; Mueller, Anna; Schaberle, Till F.; Hughes, Dallas E.; Epstein, Slava; Jones, Michael; Lazarides, Linos; Steadman, Victoria A.; Cohen, Douglas R.; Felix, Cintia R.; Fetterman, K. Ashley; Millett, William P.; Nitti, Anthony G.; Zullo, Ashley M.; Chen, Chao; Lewis, Kim
      Nature (London, United Kingdom) (2015), 517 (7535), 455-459CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Antibiotic resistance is spreading faster than the introduction of new compds. into clin. practice, causing a public health crisis. Most antibiotics were produced by screening soil microorganisms, but this limited resource of cultivable bacteria was overmined by the 1960s. Synthetic approaches to produce antibiotics have been unable to replace this platform. Uncultured bacteria make up approx. 99% of all species in external environments, and are an untapped source of new antibiotics. We developed several methods to grow uncultured organisms by cultivation in situ or by using specific growth factors. Here we report a new antibiotic that we term teixobactin, discovered in a screen of uncultured bacteria. Teixobactin inhibits cell wall synthesis by binding to a highly conserved motif of lipid II (precursor of peptidoglycan) and lipid III (precursor of cell wall teichoic acid). We did not obtain any mutants of Staphylococcus aureus or Mycobacterium tuberculosis resistant to teixobactin. The properties of this compd. suggest a path towards developing antibiotics that are likely to avoid development of resistance.
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      Mode of action of teixobactins in cellular membranes
      Shukla, Rhythm; Medeiros-Silva, Joao; Parmar, Anish; Vermeulen, Bram J. A.; Das, Sanjit; Paioni, Alessandra Lucini; Jekhmane, Shehrazade; Lorent, Joseph; Bonvin, Alexandre M. J. J.; Baldus, Marc; Lelli, Moreno; Veldhuizen, Edwin J. A.; Breukink, Eefjan; Singh, Ishwar; Weingarth, Markus
      Nature Communications (2020), 11 (1), 2848CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      The natural antibiotic teixobactin kills pathogenic bacteria without detectable resistance. The difficult synthesis and unfavorable soly. of teixobactin require modifications, yet insufficient knowledge on its binding mode impedes the hunt for superior analogs. Thus far, teixobactins are assumed to kill bacteria by binding to cognate cell wall precursors (Lipid II and III). Here we present the binding mode of teixobactins in cellular membranes using solid-state NMR, microscopy, and affinity assays. We solve the structure of the complex formed by an improved teixobactin-analog and Lipid II and reveal how teixobactins recognize a broad spectrum of targets. Unexpectedly, we find that teixobactins only weakly bind to Lipid II in cellular membranes, implying the direct interaction with cell wall precursors is not the sole killing mechanism. Our data suggest an addnl. mechanism affords the excellent activity of teixobactins, which can block the cell wall biosynthesis by capturing precursors in massive clusters on membranes.
    3. 3
      Shukla, R.; Lavore, F.; Maity, S.; Derks, M. G. N.; Jones, C. R.; Vermeulen, B. J. A.; Melcrová, A.; Morris, M. A.; Becker, L. M.; Wang, X.; Kumar, R.; Medeiros-Silva, J.; van Beekveld, R. A. M.; Bonvin, A. M. J. J.; Lorent, J. H.; Lelli, M.; Nowick, J. S.; MacGillavry, H. D.; Peoples, A. J.; Spoering, A. L.; Ling, L. L.; Hughes, D. E.; Roos, W. H.; Breukink, E.; Lewis, K.; Weingarth, M. Teixobactin kills bacteria by a two-pronged attack on the cell envelope. Nature 2022, 608, 390– 396,  DOI: 10.1038/s41586-022-05019-y
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      Teixobactin kills bacteria by a two-pronged attack on the cell envelope
      Shukla, Rhythm; Lavore, Francesca; Maity, Sourav; Derks, Maik G. N.; Jones, Chelsea R.; Vermeulen, Bram J. A.; Melcrova, Adela; Morris, Michael A.; Becker, Lea Marie; Wang, Xiaoqi; Kumar, Raj; Medeiros-Silva, Joao; van Beekveld, Roy A. M.; Bonvin, Alexandre M. J. J.; Lorent, Joseph H.; Lelli, Moreno; Nowick, James S.; MacGillavry, Harold D.; Peoples, Aaron J.; Spoering, Amy L.; Ling, Losee L.; Hughes, Dallas E.; Roos, Wouter H.; Breukink, Eefjan; Lewis, Kim; Weingarth, Markus
      Nature (London, United Kingdom) (2022), 608 (7922), 390-396CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)
      Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1-3. Teixobactin4 represents a new class of antibiotics with a unique chem. scaffold and lack of detectable resistance. Teixobactin targets lipid II , a precursor of peptidoglycan5. Here the authors unravel the mechanism of teixobactin at the at. level using a combination of solid-state NMR, microscopy, in vivo assays and mol. dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II , whereas the N terminus coordinates the pyrophosphate of another lipid II mol. This configuration favors the formation of a β-sheet of teixobactins bound to the target, creating a supramol. fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramol. structure compromises membrane integrity. Atomic force microscopy and mol. dynamics simulations show that the supramol. structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concd. within the supramol. structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II , which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compd. targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.
    4. 4
      Yang, H.; Wierzbicki, M.; Du Bois, D. R.; Nowick, J. S. X-ray Crystallographic Structure of a Teixobactin Derivative Reveals Amyloid-like Assembly. J. Am. Chem. Soc. 2018, 140, 14028– 14032,  DOI: 10.1021/jacs.8b07709
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      X-ray Crystallographic Structure of a Teixobactin Derivative Reveals Amyloid-like Assembly
      Yang, Hyunjun; Wierzbicki, Michal; Du Bois, Derek R.; Nowick, James S.
      Journal of the American Chemical Society (2018), 140 (43), 14028-14032CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      This paper describes the X-ray crystallog. structure of a deriv. of the antibiotic teixobactin and shows that its supramol. assembly through the formation of antiparallel β-sheets creates binding sites for oxyanions. An active deriv. of teixobactin contg. lysine in place of allo-enduracididine assembles to form amyloid-like fibrils, which are obsd. through a thioflavin T fluorescence assay and by transmission electron microscopy. A homolog, bearing an N-Me substituent, to attenuate fibril formation, and an iodine atom, to facilitate X-ray crystallog. phase detn., crystallizes as double helixes of β-sheets that bind sulfate anions. β-Sheet dimers are key subunits of these assemblies, with the N-terminal methylammonium group of one monomer and the C-terminal macrocycle of the other monomer binding each anion. These observations suggest a working model for the mechanism of action of teixobactin, in which the antibiotic assembles and the assemblies bind lipid II and related bacterial cell wall precursors on the surface of Gram-pos. bacteria.
    5. 5
      Yang, H.; Pishenko, A. V.; Li, X.; Nowick, J. S. Design, Synthesis, and Study of Lactam and Ring-Expanded Analogues of Teixobactin. J. Org. Chem. 2020, 85, 1331– 1339,  DOI: 10.1021/acs.joc.9b02631
      5
      Design, synthesis, and study of lactam and ring-expanded analogs of teixobactin
      Yang, Hyunjun; Pishenko, Arthur V.; Li, Xingyue; Nowick, James S.
      Journal of Organic Chemistry (2020), 85 (3), 1331-1339CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      This paper describes the chem. synthesis, X-ray crystallog. structure, and antibiotic activity assay of lactam analogs of teixobactin and explores ring-expanded analogs of teixobactin with β3-homo amino acids. Lactam analogs of teixobactin contg. all four stereoisomers of aza-threonine at position 8 were synthesized on a solid support from com. available stereoisomeric threonine derivs. The threonine stereoisomers are converted to the diastereomeric aza-threonines by mesylation, azide displacement, and redn. during the synthesis. D-Aza-Thr8,Arg10-teixobactin exhibits 2-8-fold greater antibiotic activity than the corresponding macrolactone Arg10-teixobactin. Azateixobactin analogs contg. other stereoisomers of aza-threonine are inactive. A dramatic 16-128-fold increase in the activity of teixobactin and teixobactin analogs is obsd. with the inclusion of 0.002% of the mild detergent polysorbate 80 in the MIC assay. The X-ray crystallog. structure of N-Me-D-Gln4,D-aza-Thr8,Arg10-teixobactin reveals an amphipathic hydrogen-bonded antiparallel β-sheet dimer that binds chloride anions. In the binding site, the macrolactam amide NH groups of residues 8, 10, and 11, as well as the extra amide NH group of the lactam ring, hydrogen bond to the chloride anion. The teixobactin pharmacophore tolerates ring expansion of the 13-membered ring to 14-,15-, and 16-membered rings contg. β3-homo amino acids with retention of partial or full antibiotic activity.
    6. 6
      Yang, H.; Du Bois, D. R.; Ziller, J. W.; Nowick, J. S. X-ray crystallographic structure of a teixobactin analogue reveals key interactions of the teixobactin pharmacophore. Chem. Commun. 2017, 53, 2772– 2775,  DOI: 10.1039/C7CC00783C
      6
      X-ray crystallographic structure of a teixobactin analogue reveals key interactions of the teixobactin pharmacophore
      Yang, H.; Du Bois, D. R.; Ziller, J. W.; Nowick, J. S.
      Chemical Communications (Cambridge, United Kingdom) (2017), 53 (18), 2772-2775CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      The x-ray crystallog. structure of a truncated teixobactin analog reveals hydrogen-bonding and hydrophobic interactions and a cavity that binds a chloride anion. Min. inhibitory concn. (MIC) assays against Gram-pos. bacteria correlate the obsd. structure with antibiotic activity.
    7. 7
      Yang, H.; Chen, K. H.; Nowick, J. S. Elucidation of the Teixobactin Pharmacophore. ACS Chem. Biol. 2016, 11, 1823– 1826,  DOI: 10.1021/acschembio.6b00295
      7
      Elucidation of the Teixobactin Pharmacophore
      Yang, Hyunjun; Chen, Kevin H.; Nowick, James S.
      ACS Chemical Biology (2016), 11 (7), 1823-1826CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      This paper elucidates the teixobactin pharmacophore by comparing the arginine analog of teixobactin Arg10-teixobactin to seven homologues with varying structure and stereochem. The roles of the guanidinium group at position 10, the stereochem. of the macrolactone ring, and the "tail" comprising residues 1-5 are investigated. The guanidinium group is not necessary for activity; Lys10-teixobactin is more active than Arg10-teixobactin against Gram-pos. bacteria in min. inhibitory concn. (MIC) assays. The relative stereochem. of the macrolactone ring is important. Diastereomer L-Thr8,Arg10-teixobactin is inactive, and diastereomer D-allo-Ile11,Arg10-teixobactin is less active. The macrolactone ring is crit.; seco-Arg10-teixobactin is inactive. The abs. stereochem. is not important; the enantiomer ent-Arg10-teixobactin is comparable in activity. The hydrophobic N-terminal tail is important. Truncation of residues 1-5 results in loss of activity, and replacement of residues 1-5 with a dodecanoyl group partially restores activity. These findings pave the way for developing simpler homologues of teixobactin with enhanced pharmacol. properties.
    8. 8
      Kabsch, W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125– 132,  DOI: 10.1107/S0907444909047337
      8
      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.
    9. 9
      Foadi, J.; Aller, P.; Alguel, Y.; Cameron, A.; Axford, D.; Owen, R. L.; Armour, W.; Waterman, D. G.; Iwata, S.; Evans, G. Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2013, 69, 1617– 1632,  DOI: 10.1107/S0907444913012274
      9
      Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography
      Foadi, James; Aller, Pierre; Alguel, Yilmaz; Cameron, Alex; Axford, Danny; Owen, Robin L.; Armour, Wes; Waterman, David G.; Iwata, So; Evans, Gwyndaf
      Acta Crystallographica, Section D: Biological Crystallography (2013), 69 (8), 1617-1632CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      The availability of intense microbeam macromol. crystallog. beamlines at third-generation synchrotron sources has enabled data collection and structure soln. from microcrystals of <10 μm in size. The increased likelihood of severe radiation damage where microcrystals or particularly sensitive crystals are used forces crystallographers to acquire large nos. of data sets from many crystals of the same protein structure. The assocd. anal. and merging of multi-crystal data is currently a manual and time-consuming step. Here, a computer program, BLEND, that has been written to assist with and automate many of the steps in this process is described. It is demonstrated how BLEND has successfully been used in the soln. of a novel membrane protein.
    10. 10
      Grosse-Kunstleve, R. W.; Adams, P. D. Substructure search procedures for macromolecular structures. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2003, 59, 1966– 1973,  DOI: 10.1107/S0907444903018043
      10
      Substructure search procedures for macromolecular structures
      Grosse-Kunstleve, R. W.; Adams, P. D.
      Acta Crystallographica, Section D: Biological Crystallography (2003), D59 (11), 1966-1973CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
      This paper accompanies a lecture given at the 2003 CCP4 Study Weekend on exptl. phasing. The first part is an overview of the fundamentals of Patterson methods and direct methods with the audience of the CCP4 Study Weekend in mind. In the second part, a new hybrid substructure search is outlined.
    11. 11
      Adams, P. D.; Afonine, P. V.; Bunkoczi, 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– 221,  DOI: 10.1107/S0907444909052925
      11
      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.
    12. 12
      Terwilliger, T. C.; Adams, P. D.; Read, R. J.; McCoy, A. J.; Moriarty, N. W.; Grosse-Kunstleve, R. W.; Afonine, P. V.; Zwart, P. H.; Hung, L. W. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65, 582– 601,  DOI: 10.1107/S0907444909012098
      12
      Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard
      Terwilliger, Thomas C.; Adams, Paul D.; Read, Randy J.; McCoy, Airlie J.; Moriarty, Nigel W.; Grosse-Kunstleve, Ralf W.; Afonine, Pavel V.; Zwart, Peter H.; Hung, Li Wei
      Acta Crystallographica, Section D: Biological Crystallography (2009), 65 (6), 582-601CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      Ests. of the quality of exptl. maps are important in many stages of structure detn. of macromols. Map quality is defined here as the correlation between a map and the corresponding map obtained using phases from the final refined model. Here, ten different measures of exptl. map quality were examd. using a set of 1359 maps calcd. by re-anal. of 246 solved MAD, SAD and MIR data sets. A simple Bayesian approach to estn. of map quality from one or more measures is presented. It was found that a Bayesian estimator based on the skewness of the d. values in an electron-d. map is the most accurate of the ten individual Bayesian estimators of map quality examd., with a correlation between estd. and actual map quality of 0.90. A combination of the skewness of electron d. with the local correlation of r.m.s. d. gives a further improvement in estg. map quality, with an overall correlation coeff. of 0.92. The PHENIX AutoSol wizard carries out automated structure soln. based on any combination of SAD, MAD, SIR or MIR data sets. The wizard is based on tools from the PHENIX package and uses the Bayesian ests. of map quality described here to choose the highest quality solns. after exptl. phasing.
    13. 13
      Afonine, P. V.; Grosse-Kunstleve, R. W.; Echols, N.; Headd, J. J.; Moriarty, N. W.; Mustyakimov, M.; Terwilliger, T. C.; Urzhumtsev, A.; Zwart, P. H.; Adams, P. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2012, 68, 352– 367,  DOI: 10.1107/S0907444912001308
      13
      Towards automated crystallographic structure refinement with phenix.refine
      Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel; Headd, Jeffrey J.; Moriarty, Nigel W.; Mustyakimov, Marat; Terwilliger, Thomas C.; Urzhumtsev, Alexandre; Zwart, Peter H.; Adams, Paul D.
      Acta Crystallographica, Section D: Biological Crystallography (2012), 68 (4), 352-367CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      Phenix.refine is a program within the PHENIX package that supports crystallog. structure refinement against exptl. data with a wide range of upper resoln. limits using a large repertoire of model parameterizations. It has several automation features and is also highly flexible. Several hundred parameters enable extensive customizations for complex use cases. Multiple user-defined refinement strategies can be applied to specific parts of the model in a single refinement run. An intuitive graphical user interface is available to guide novice users and to assist advanced users in managing refinement projects. X-ray or neutron diffraction data can be used sep. or jointly in refinement. phenix.refine is tightly integrated into the PHENIX suite, where it serves as a crit. component in automated model building, final structure refinement, structure validation and deposition to the wwPDB. This paper presents an overview of the major phenix.refine features, with extensive literature refs. for readers interested in more detailed discussions of the methods.
    14. 14
      Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486– 501,  DOI: 10.1107/S0907444910007493
      14
      Features and development of Coot
      Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
    15. 15
      Moriarty, N. W.; Grosse-Kunstleve, R. W.; Adams, P. D. electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65, 1074– 1080,  DOI: 10.1107/S0907444909029436
      15
      electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation
      Moriarty, Nigel W.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
      Acta Crystallographica, Section D: Biological Crystallography (2009), 65 (10), 1074-1080CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      The electronic Ligand Builder and Optimization Workbench (eLBOW) is a program module of the PHENIX suite of computational crystallog. software. It is designed to be a flexible procedure that uses simple and fast quantum-chem. techniques to provide chem. accurate information for novel and known ligands alike. A variety of input formats and options allow the attainment of a no. of diverse goals including geometry optimization and generation of restraints.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.joc.3c02617.

    • HPLC and MS characterization data and X-ray crystallographic statistics for N-Me-d-Gln4,Lys10-teixobactin (1) (PDF)

    Accession Codes

    Crystallographic coordinates of N-Me-d-Gln4,Lys10-teixobactin (1) were deposited into the Protein Data Bank (PDB) with code 8U78.


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