Abstract
Bacillus anthracis is a spore-forming gram-positive bacterium responsible for anthrax, an infectious disease with a high mortality rate and a target of concern due to bioterrorism and long-term site contamination. The entire surface of vegetative cells in exponential or stationary growth phase is covered in proteinaceous arrays called S-layers, composed of Sap or EA1 protein, respectively. The Sap S-layer represents an important virulence factor and cell envelope support structure whose paracrystalline nature is essential for its function. However, the spatial organization of Sap in its lattice state remains elusive. Here, we employed cryoelectron tomography and subtomogram averaging to obtain a map of the Sap S-layer from tubular polymers that revealed a conformational switch between the postassembly protomers and the previously available X-ray structure of the condensed monomers. To build and validate an atomic model of the lattice within this map, we used a combination of molecular dynamics simulations, X-ray crystallography, cross-linking mass spectrometry, and biophysics in an integrative structural biology approach. The Sap lattice model produced recapitulates a close-to-physiological arrangement, reveals high-resolution details of lattice contacts, and sheds light on the mechanisms underlying the stability of the Sap layer.
| Original language | English |
|---|---|
| Article number | e2415351121 |
| Number of pages | 11 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 121 |
| Issue number | 51 |
| DOIs | |
| Publication status | Published - 17 Dec 2024 |
Bibliographical note
Funding Information:This work was partially supported by Vlaams Instituut voor Biotechnologie (VIB) and Fonds voor Wetenschappelijk Onderzoek (FWO) Flanders through project Grant No. G065220N. A.F. was supported by a postdoctoral fellowship from FWO (1253121N). A.S. was supported by the European Molecular Biology Organization (EMBO-ALTF-709-2021) and the Marie Sk\u0142odowska-Curie Actions (MSCA) MSCA-SLYDIV project. K.L. and M.K. were funded by the Sofja Kovalevskaja Award from the Alexander von Humboldt Foundation. M.K. is supported by the Heisenberg Award from Deutsche Forschungsgemeinschaft and by the Helmholtz Society. E.V.H., A.J.C., and A.J.P. were supported by the National Institute of Allergy and Infectious Diseases of the NIH under Award No. R21AI168838. We thank Bio Electron Cryogenic Microscopy (BECM) and Dr. Marcus Fislage for his assistance during cryo-EM data collection. We thank the staff of the synchrotron SOLEIL and Diamond for assistance and support in using their beamlines. We thank Deryck Mills for his assistance with (and assiduous maintenance of) the microscopes as well as Dr. \u00D6zkan Yildiz and Dr. Juan Castillo-Hernandez for their Information Technology support at the Max Planck Institute of Biophysics. We acknowledge the use of high-performance computing resources from the Cyber Infrastructure & Advanced Research Computing Group at Colorado School of Mines and through allocation BIO220015 from the Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support program, which is supported by NSF Grants #2138259, #2138286, #2138307, #2137603, and #2138296.
Publisher Copyright:
Copyright © 2024 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
Keywords
- Bacillus anthracis/metabolism
- Bacterial Proteins/chemistry
- Molecular Dynamics Simulation
- Crystallography, X-Ray
- Cryoelectron Microscopy/methods
- Membrane Glycoproteins/metabolism
- Protein Conformation
- Models, Molecular