Characterization Of Intermolecular Quaternary Interactions Between Discrete Segments Of The Streptococcus Mutans Adhesin P1 And Their Binding To Small Molecule Amyloid Inhibitors Via Nmr Spectroscopy

Cell surface-localized P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans mediates sucrose-independent adhesion to tooth surfaces. Previous studies showed that P1's C-terminal segment (C123, AgII) is also liberated as a separate polypepeptide, contributes to cellular adhesion, interacts specifically with intact P1 on the cell surface, and forms amyloid fibrils. Identifying how C123 specifically interacts with P1 at the atomic level is essential for understanding related virulence properties of S. mutans. Characterizing its interactions with small molecule amyloid inhibitors may provide an avenue for inhibiting biofilm formation. Using NMR spectroscopy, we characterized the biologically relevant interactions of the individual C3 domain with A3VP1, a polypeptide that represents the apical head of P1 as it is projected on the cell surface. We also evaluated C3's interaction with C12 and the adhesion-inhibiting monoclonal antibody (MAb) 6-8C. NMR titration experiments with 15N-enriched C3 demonstrate its specific binding to A3VP1. Based on resolved C3 assignments, two binding sites, proximal and distal, are identified. Complementary NMR titration of A3VP1 with a C3/C12 complex suggests that binding of A3VP1 occurs on the distal C3 binding site, while the proximal site is occupied by C12. The MAb 6-8C binding interface to C3 overlaps with that of A3VP1 at the distal site. Together these results identify a specific C3-A3VP1interaction that serves as a foundation for understanding the interaction of C123 with P1 on the bacterial surface and the related biological processes that stem from this interaction. Additionally, we demonstrate the interaction of epigallocatechin-3-gallate (EGCG) and resveratrol (RSV) with C123 and C3 domains at physiologic pH by WaterLOGSY NMR experiments.