Staphylococcus aureus secretes a family of serine protease–like enzymes (SplA to SplF) that resemble eukaryotic granzymes, yet the mechanism by which amino-terminal processing activates this subclass has remained unresolved. Structural studies show insertion of the processed amino terminus without detectable changes in active-site geometry, creating a longstanding paradox as to how catalytic competence is achieved. Here, we identify SplB as the most highly expressed member of this family in a pathogenic methicillin-resistant S. aureus strain and use it to define the basis of activation. Solution nuclear magnetic resonance spectroscopy shows that precise amino-terminal processing triggers a long-range allosteric network coupling the amino terminus to the active site ∼20 angstroms away, unlocking global microsecond-to-millisecond dynamics that enable substrate engagement. Molecular dynamics simulations reveal the conformational ensembles underlying these motions. Last, mutational perturbation of this dynamic network modulates substrate engagement and catalytic activity in a manner consistent with dynamic control of binding competence. Together, these findings establish dynamic allostery as the mechanism of N-terminal activation in this subclass of serine proteases.
Eunjeong Lee, J. Redzic, Samrat Sarkar et al.· Science Advances· 0 citations
Sortase A (SrtA) enzymes covalently anchor surface proteins to Gram-positive bacterial cell walls, promoting colonization and virulence. In Streptococcus pneumoniae, previous studies identified both a domain-swapped dimer and an active refolded monomer, but the active enzyme has not been characterized at the structural and residue-specific level. Here, we performed quantitative proteomic comparisons of wild-type and SrtA knockout strains that confirmed the loss of multiple LPxTG-containing virulence factors, including ZmpB, NanA, and IgA1 protease, consistent with an essential role for SrtA in surface protein anchoring. To enable mechanistic studies, we established a biochemical framework to produce monomeric Streptococcus pneumoniae SrtA by refolding and developed a gel-based assay using recombinant substrates to monitor catalytic activity. The refolded monomer, but not the swapped dimer, catalyzed cleavage and transpeptidation of a canonical LPxTG substrate in a metal-independent manner under the conditions examined. We further report high-resolution NMR backbone assignments for the active monomer and identify substrate-induced chemical shift perturbations that localize to the active site. Together, these findings provide an integrated proteomic, biochemical, and NMR characterization of monomeric, catalytically active Streptococcus pneumoniae SrtA and reveal residue-specific interactions with a canonical LPNTG recognition peptide.
Eunjeong Lee, Blaine H. Gordon, J. Redzic et al.· Biomolecules· 0 citations