Recent advances in iterative neural network analyses (e.g., AlphaFold2 and RoseTTA fold) have been revolutionary for protein 3D structure prediction, especially for difficult-to-manipulate α-helical/β-barrel integral membrane proteins. These model structures are calculated based on the coevolution of amino acids within the protein of interest and similarities to existing protein structures; the local effects of the membrane on folding and stability of the calculated model structures are not considered. We recently reported the discovery, 3D modeling, and characterization of 18-β-stranded outer-membrane (OM) WzpX, WzpS, and WzpB β-barrel secretion porins for the exopolysaccharide (EPS), major spore coat polysaccharide (MASC), and biosurfactant polysaccharide (BPS) pathways (respectively) in the Gram-negative social predatory bacterium Myxococcus xanthus DZ2. However, information was not obtained regarding the dynamic behavior of surface-gating WzpX/S/B loop domains or on potential treatments to inactivate these porins. Herein, we developed a molecular dynamics (MD) protocol to study the core stability and loop dynamism of neural network-based integral membrane protein structure models embedded in an asymmetric OM bilayer, using the M. xanthus WzpX, WzpS, and WzpB proteins as test candidates. This was accomplished through integration of the CHARMM-graphical user interface (GUI) and Molecular Operating Environment (MOE) workflows to allow for a rapid simulation system setup and facilitate data analysis. In addition to serving as a method of model structure validation, our molecular dynamics simulations revealed a minimal movement of extracellular WzpX/S/B loops in the absence of an external stimulus as well as druggable cavities between the loops. Virtual screening of a commercial fragment library against these cavities revealed putative fragment-binding hotspots on the cell-surface face of each β-barrel, along with key interacting residues, and identified promising hits for the design of potential binders capable of plugging the β-barrels and inhibiting polysaccharide secretion.

Chemical Computing Group Montreal Quebec H3A 2R7 Canada

Department of Chemistry Faculty of Science University of Hradec Kralove Rokitanskeho 62 50003 Hradec Kralove Czech Republic

Institut National de la Recherche Scientifique Centre Armand Frappier Santé Biotechnologie Université du Québec Institut Pasteur International Network Laval QC H7V 1B7 Canada

Laboratory of Molecular Modeling Applied to Chemical and Biological Defense Military Institute of Engineering Rio de Janeiro 22290 270 Brazil

PROTEO the Quebec Network for Research on Protein Function Engineering and Applications Université Laval Quebec QC G1V 0A6 Canada

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Jumper J.; Evans R.; Pritzel A.; Green T.; Figurnov M.; Ronneberger O.; Tunyasuvunakool K.; Bates R.; Žídek A.; Potapenko A.; et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021, 596 (7873), 583–589. 10.1038/s41586-021-03819-2. PubMed DOI PMC

Baek M.; DiMaio F.; Anishchenko I.; Dauparas J.; Ovchinnikov S.; Lee G. R.; Wang J.; Cong Q.; Kinch L. N.; Schaeffer R. D.; et al. Accurate prediction of protein structures and interactions using a three-track neural network. Science 2021, 373 (6557), 871–876. 10.1126/science.abj8754. PubMed DOI PMC

Islam S. T.; Lam J. S. Topological mapping methods for α-helical bacterial membrane proteins – an update and a guide. MicrobiologyOpen 2013, 2 (2), 350–364. 10.1002/mbo3.72. PubMed DOI PMC

Gilmore R.; Mandon E. C. Understanding integration of α-helical membrane proteins: the next steps. Trends Biochem. Sci. 2012, 37 (8), 303–308. 10.1016/j.tibs.2012.05.003. PubMed DOI PMC

Schleiff E.; Soll J. Membrane protein insertion: mixing eukaryotic and prokaryotic concepts. EMBO Rep. 2005, 6 (11), 1023–1027. 10.1038/sj.embor.7400563. PubMed DOI PMC

Chavent M.; Duncan A. L.; Sansom M. S. P. Molecular dynamics simulations of membrane proteins and their interactions: from nanoscale to mesoscale. Curr. Opin. Struct. Biol. 2016, 40, 8–16. 10.1016/j.sbi.2016.06.007. PubMed DOI PMC

Saïdi F.; Mahanta U.; Panda A.; Kezzo A. A.; Jolivet N. Y.; Bitazar R.; John G.; Martinez M.; Mellouk A.; Calmettes C.; et al. Bacterial outer membrane polysaccharide export (OPX) proteins occupy three structural classes with selective β-barrel porin requirements for polymer secretion. Microbiol. Spectr. 2022, 10 (5), e01290-22.10.1128/spectrum.01290-22. PubMed DOI PMC

Islam S. T.; Vergara Alvarez I.; Saïdi F.; Guiseppi A.; Vinogradov E.; Sharma G.; Espinosa L.; Morrone C.; Brasseur G.; Guillemot J.-F.; et al. Modulation of bacterial multicellularity via spatio-specific polysaccharide secretion. PLOS Biol. 2020, 18, e3000728.10.1371/journal.pbio.3000728. PubMed DOI PMC

Saïdi F.; Gamboa Marin O. J.; Veytia-Bucheli J. I.; Vinogradov E.; Ravicoularamin G.; Jolivet N. Y.; Kezzo A. A.; Ramirez Esquivel E.; Panda A.; Sharma G.; et al. Evaluation of azido 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) analogues for click chemistry-mediated metabolic labeling of Myxococcus xanthus DZ2 lipopolysaccharide. ACS Omega 2022, 7 (39), 34997–35013. 10.1021/acsomega.2c03711. PubMed DOI PMC

Muñoz-Dorado J.; Marcos-Torres F. J.; García-Bravo E.; Moraleda-Muñoz A.; Pérez J. Myxobacteria: moving, killing, feeding, and surviving together. Front. Microbiol. 2016, 7, 781.10.3389/fmicb.2016.00781. PubMed DOI PMC

Faure L. M.; Fiche J.-B.; Espinosa L.; Ducret A.; Anantharaman V.; Luciano J.; Lhospice S.; Islam S. T.; Tréguier J.; Sotes M.; et al. The mechanism of force transmission at bacterial focal adhesion complexes. Nature 2016, 539 (7630), 530–535. 10.1038/nature20121. PubMed DOI PMC

Jolivet N. Y.; Han E.; Belgrave A. M.; Saïdi F.; Koushki N.; Lemon D. J.; Faure L. M.; Fleuchot B.; Mahanta U.; Jiang H. Integrin-like adhesin CglD confers traction and stabilizes bacterial focal adhesions involved in myxobacterial gliding motility. bioRxiv 2023, 1–41. 10.1101/2023.10.19.562135. DOI

Islam S. T.; Jolivet N. Y.; Cuzin C.; Belgrave A. M.; My L.; Fleuchot B.; Faure L. M.; Mahanta U.; Kezzo A. A.; Saïdi F.; et al. Unmasking of the von Willebrand A-domain surface adhesin CglB at bacterial focal adhesions mediates myxobacterial gliding motility. Sci. Adv. 2023, 9, eabq061910.1126/sciadv.abq0619. PubMed DOI PMC

Saïdi F.; Bitazar R.; Bradette N.; Islam S. T. Bacterial glycocalyx integrity impacts tolerance of Myxococcus xanthus to antibiotics and oxidative-stress agents. Biomolecules 2022, 12 (4), 571.10.3390/biom12040571. PubMed DOI PMC

Saïdi F.; Jolivet N. Y.; Lemon D. J.; Nakamura A.; Belgrave A. M.; Garza A. G.; Veyrier F. J.; Islam S. T. Bacterial glycocalyx integrity drives multicellular swarm biofilm dynamism. Mol. Microbiol. 2021, 116 (4), 1151–1172. 10.1111/mmi.14803. PubMed DOI

Schwabe J.; Pérez-Burgos M.; Herfurth M.; Glatter T.; So̷gaard-Andersen L. Evidence for a widespread third system for bacterial polysaccharide export across the outer membrane comprising a composite OPX/β-barrel translocon. mBio 2022, 13, 5.10.1128/mbio.02032-22. PubMed DOI PMC

Pérez-Burgos M.; García-Romero I.; Jung J.; Schander E.; Valvano M. A.; So̷gaard-Andersen L. Characterization of the exopolysaccharide biosynthesis pathway in Myxococcus xanthus. J. Bacteriol. 2020, 202, e00335-20.10.1128/jb.00335-20. PubMed DOI PMC

Islam S. T.; Lam J. S. Synthesis of bacterial polysaccharides via the Wzx/Wzy-dependent pathway. Can. J. Microbiol. 2014, 60 (11), 697–716. 10.1139/cjm-2014-0595. PubMed DOI

Islam S. T.; Taylor V. L.; Qi M.; Lam J. S. Membrane topology mapping of the O-antigen flippase (Wzx), polymerase (Wzy), and ligase (WaaL) from Pseudomonas aeruginosa PAO1 reveals novel domain architectures. mBio 2010, 1 (3), e00189-10.10.1128/mBio.00189-10. PubMed DOI PMC

Islam S. T.; Eckford P. D. W.; Jones M. L.; Nugent T.; Bear C. E.; Vogel C.; Lam J. S. Proton-dependent gating and proton uptake by Wzx support O-antigen-subunit antiport across the bacterial inner membrane. mBio 2013, 4 (5), e00678-1310.1128/mBio.00678-13. PubMed DOI PMC

Islam S. T.; Lam J. S. Wzx flippase-mediated membrane translocation of sugar polymer precursors in bacteria. Environ. Microbiol. 2013, 15 (4), 1001–1015. 10.1111/j.1462-2920.2012.02890.x. PubMed DOI

Islam S. T.; Gold A. C.; Taylor V. L.; Anderson E. M.; Ford R. C.; Lam J. S. Dual conserved periplasmic loops possess essential charge characteristics that support a catch-and-release mechanism of O-antigen polymerization by Wzy in Pseudomonas aeruginosa PAO1. J. Biol. Chem. 2011, 286 (23), 20600–20605. 10.1074/jbc.C110.204651. PubMed DOI PMC

Islam S. T.; Huszczynski S. M.; Nugent T.; Gold A. C.; Lam J. S. Conserved-residue mutations in Wzy affect O-antigen polymerization and Wzz-mediated chain-length regulation in Pseudomonas aeruginosa PAO1. Sci. Rep 2013, 3, 3441.10.1038/srep03441. PubMed DOI PMC

Whitney J. C.; Howell P. L. Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria. Trends Microbiol. 2013, 21 (2), 63–72. 10.1016/j.tim.2012.10.001. PubMed DOI PMC

Molecular Operating Environment (MOE); Chemical Computing Group ULC: Montreal, QC, Canada, 2023. https://www.chemcomp.com/Products.htm

Brooks B. R.; Bruccoleri R. E.; Olafson B. D.; States D. J.; Swaminathan S.; Karplus M. CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 1983, 4 (2), 187–217. 10.1002/jcc.540040211. DOI

Jo S.; Kim T.; Iyer V. G.; Im W. CHARMM-GUI: A web-based graphical user interface for CHARMM. J. Comput. Chem. 2008, 29 (11), 1859–1865. 10.1002/jcc.20945. PubMed DOI

Wang Y.; Andole Pannuri A.; Ni D.; Zhou H.; Cao X.; Lu X.; Romeo T.; Huang Y. Structural basis for translocation of a biofilm-supporting exopolysaccharide across the bacterial outer membrane*. J. Biol. Chem. 2016, 291 (19), 10046–10057. 10.1074/jbc.M115.711762. PubMed DOI PMC

Wu Emilia L.; Fleming Patrick J.; Yeom Min S.; Widmalm G.; Klauda Jeffery B.; Fleming Karen G.; Im W. E. coli outer membrane and interactions with OmpLA. Biophys. J. 2014, 106 (11), 2493–2502. 10.1016/j.bpj.2014.04.024. PubMed DOI PMC

Dowhan W. Molecular basis for membrane phospholipid diversity: why are there so many lipids?. Annu. Rev. Biochem. 1997, 66 (1), 199–232. 10.1146/annurev.biochem.66.1.199. PubMed DOI

MacLean L.; Perry M. B.; Nossova L.; Kaplan H.; Vinogradov E. The structure of the carbohydrate backbone of the LPS from Myxococcus xanthus strain DK1622. Carbohydr. Res. 2007, 342 (16), 2474–2480. 10.1016/j.carres.2007.07.023. PubMed DOI

Holkenbrink C.; Hoiczyk E.; Kahnt J.; Higgs P. I. Synthesis and assembly of a novel glycan layer in Myxococcus xanthus spores. J. Biol. Chem. 2014, 289, 32364.10.1074/jbc.M114.595504. PubMed DOI PMC

Jakobczak B.; Keilberg D.; Wuichet K.; So̷gaard-Andersen L. Contact- and protein transfer-dependent stimulation of assembly of the gliding motility machinery in Myxococcus xanthus. PLOS Genet. 2015, 11 (7), e100534110.1371/journal.pgen.1005341. PubMed DOI PMC

França T. C. C.; Botelho F. D.; Drummond M. L.; LaPlante S. R. Theoretical investigation of repurposed drugs potentially capable of binding to the catalytic site and the secondary binding pocket of subunit A of ricin. ACS Omega 2022, 7 (36), 32805–32815. 10.1021/acsomega.2c04819. PubMed DOI PMC

Ali Z.; Cardoza J. V.; Basak S.; Narsaria U.; Singh V. P.; Isaac S. P.; França T. C. C.; LaPlante S. R.; George S. S. Computational design of candidate multi-epitope vaccine against SARS-CoV-2 targeting structural (S and N) and non-structural (NSP3 and NSP12) proteins. J. Biomol. Struct. Dyn. 2023, 1–20. 10.1080/07391102.2023.2173297. PubMed DOI

Case D. A.; Darden T. A.; Cheatham T. E. III; Simmerling C. L.; Wang J.; Duke R. E.; Luo R.; Crowley M.; Walker R. C.; Zhang W.. et al.AMBER 10. University of California: San Francisco, 2008.

Nelson M. T.; Humphrey W.; Gursoy A.; Dalke A.; Kalé L. V.; Skeel R. D.; Schulten K. NAMD: a parallel, object-oriented molecular dynamics program. Int. J. Supercomp. Appl. High Perform. Comput. 1996, 10 (4), 251–268. 10.1177/109434209601000401. DOI

DeLano W. L.; Bromberg S.. PyMOL User’s Guide. DeLano Scientific LLC, 2004; p. 629.

Thomsen R.; Christensen M. H. MolDock: a new technique for high-accuracy molecular docking. J. Med. Chem. 2006, 49 (11), 3315–3321. 10.1021/jm051197e. PubMed DOI

Baell J. B. Feeling nature’s PAINS: natural products, natural product drugs, and pan assay interference compounds (PAINS). J. Nat. Prod. 2016, 79 (3), 616–628. 10.1021/acs.jnatprod.5b00947. PubMed DOI

Baell J. B.; Nissink J. W. M. Seven Year Itch: Pan-Assay Interference Compounds (PAINS) in 2017— Utility and Limitations. ACS Chem. Biol. 2018, 13 (1), 36–44. 10.1021/acschembio.7b00903. PubMed DOI PMC

Huang N.; Jacobson M. P. Binding-site assessment by virtual fragment screening. PLOS One 2010, 5 (4), e1010910.1371/journal.pone.0010109. PubMed DOI PMC

Gao Y.; Widmalm G.; Im W. Modeling and simulation of bacterial outer membranes with lipopolysaccharides and capsular polysaccharides. J. Chem. Inf. Model. 2023, 63, 1592.10.1021/acs.jcim.3c00072. PubMed DOI PMC

Gao Y.; Lee J.; Widmalm G.; Im W. Modeling and simulation of bacterial outer membranes with lipopolysaccharides and enterobacterial common antigen. J. Phys. Chem. B 2020, 124 (28), 5948–5956. 10.1021/acs.jpcb.0c03353. PubMed DOI

Patel D. S.; Qi Y.; Im W. Modeling and simulation of bacterial outer membranes and interactions with membrane proteins. Curr. Opin. Struct. Biol. 2017, 43, 131–140. 10.1016/j.sbi.2017.01.003. PubMed DOI

Pavlova A.; Hwang H.; Lundquist K.; Balusek C.; Gumbart J. C. Living on the edge: simulations of bacterial outer-membrane proteins. Biochim. Biophys. Acta 2016, 1858 (7, Part B), 1753–1759. 10.1016/j.bbamem.2016.01.020. PubMed DOI