Conformational free energy landscape of sorting signal peptides in complex with bacterial sortases

Sortase enzymes are cysteine transpeptidases that anchor surface proteins to the cell wall of Gram-positive bacteria. These enzymes are widely used in protein engineering applications because they recognize and cleave a specific cell wall sorting signal on their substrates, enabling ligation to other peptides or functional molecules through sortase-mediated ligation. Among the six known sortase classes, class A sortases are the most extensively studied. However, despite detailed biochemical and structural characterization of the catalytic mechanism, the conformational dynamics during binding that positions the substrate for efficient catalysis remains less well understood. Here, we use molecular dynamics (MD) simulations combined with metadynamics to construct the conformational free energy landscape of the LPATA sorting signal peptide noncovalently bound to our recent Streptococcus pyogenes sortase A (spySrtA) crystal structure. We then compare this landscape with similar free energy surfaces obtained from MD simulations of Staphylococcus aureus sortase A (saSrtA) and Bacillus anthracis sortase B (baSrtB), each modeled with its respective sorting signal peptide substrate using AlphaFold. Our simulations reveal that substrates for spySrtA and saSrtA are dynamic and sample multiple metastable conformations distinguished by the side-chain orientation of a conserved threonine residue (referred to as Thr-In and Thr-Out orientations) that are accessible for nucleophilic attack by the catalytic cysteine. In contrast, the substrate for baSrtB exhibits reduced conformational flexibility and a stronger thermodynamic preference for a single binding pose with an inward-facing threonine orientation. We further show that amino acid substitution at the adjacent variable position (P2) on the substrate reshapes the free energy landscape, particularly affecting the flexibility of Thr-Out conformations. Together, these results highlight the importance of conformational dynamics on peptide substrate binding and how the conformational landscape modulates the dynamical equilibrium between the reactive and nonreactive conformations.