N-terminal processing unlocks global dynamics for substrate engagement in Spl proteases.

Abstract

<i>Staphylococcus aureus</i> 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 <i>S. aureus</i> 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.