A Putative Interaction between the Transmembrane Domains of Streptococcus pyogenes Sortase A and Its Endogenous Substrate M Protein Revealed by Molecular Dynamics Simulations
Year: 2025
Type: article
Abstract: Sortase enzymes are cysteine transpeptidases at the cell surface of gram-positive bacteria. Localized to distinct foci on the cell membrane, class A sortases (SrtAs) recognize a cell wall sorting signal (CWSS), and following cleavage at this specific binding motif, target proteins are ligated to precursors of the growing peptidoglycan layer. This activity of SrtA enzymes is utilized extensively in sortase-mediated ligation (SML) strategies, for a variety of protein engineering applications. Typically, engineered variants of SrtA are used for SML experiments considering the relatively low catalytic efficiency of this enzyme. Understandably, most biochemical studies are conducted with the isolated catalytic domain of SrtA enzymes from various bacteria, and the stereochemistry of the endogenous interaction between SrtA and substrate is not well understood. Here, we used AlphaFold2 to create a model of the full-length SrtA enzyme from Streptococcus pyogenes (spySrtA) with or without either a peptide substrate or a portion of M protein, a cellular target. We ran triplicate 500 ns molecular dynamics simulations for each model embedded in a lipid bilayer, which revealed several stereochemical features of this system. Contact map analyses revealed specific interactions between catalytic domain positions of spySrtA and the lipid bilayer, as well as between the enzyme and M protein residues outside the canonical LPXTG pentapeptide CWSS. We also characterized a potential transmembrane domain interaction between spySrtA and M protein that we predict orients and stabilizes substrate binding. Taken together, these interactions likely increase the catalytic efficiency of the enzyme for its substrates in vivo , and may provide important stereochemical insights for SML uses.
Institution: Western Washington University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Funders: National Science Foundation, Research Corporation for Science Advancement, National Institutes of Health, National Heart, Lung, and Blood Institute, National Institute of General Medical Sciences, Division of Chemistry, Division of Molecular and Cellular Biosciences, NHLBI Division of Intramural Research
Awards: G1508719880, G1731799598, CAREER: Bridging time and length scales of intrinsically disordered protein conformations and assemblies using advanced computational methods, RUI: A variational enhanced sampling approach to enzyme kinetics and protein dynamics in condensed phases, G475970020, G5369004629, Structural and Biochemical Studies of Blood Coagulation Factor VIII to Overcome the Immune Response, G6667359600, G6756868728, G7178219606, CAREER: The stereochemical basis of target selectivity encoded by specificity-determining loops in peptide-binding domains, Improved Understanding of Bacterial Sortase Activity Towards Next-Generation Protein Engineering, G8223309235, G8253941327
OpenAlex ID: W4417230804