Backbone NMR resonance assignments for the C terminal domain of the Streptococcus mutans adhesin P1.

Adhesin P1 (aka AgI/II) plays a pivotal role in mediating Streptococcus mutans attachment in the oral cavity, as well as in regulating biofilm development and maturation. P1's naturally occurring truncation product, Antigen II (AgII), adopts both soluble, monomeric and insoluble, amyloidogenic forms within the bacterial life cycle. Monomers are involved in important quaternary interactions that promote cell adhesion and the functional amyloid form promotes detachment of mature biofilms. The heterologous, 51-kD C123 construct comprises most of AgII and was previously characterized by X-ray crystallography. C123 contains three structurally homologous domains, C1, C2, and C3. NMR samples made using the original C123 construct, or its C3 domain, yielded moderately resolved NMR spectra. Using Alphafold, we re-analyzed the P1 sequence to better identify domain boundaries for C123, and in particular the C3 domain. We then generated a more tractable construct for NMR studies of the monomeric form, including quaternary interactions with other proteins. The addition of seven amino acids at the C-terminus greatly improved the spectral dispersion for C3 relative to the prior construct. Here we report the backbone NMR resonance assignments for the new construct and characterize some of its quaternary interactions. These data are in good agreement with the structure predicted by Alphafold, which contains additional ß-sheet secondary structure compared to the C3 domain in the C123 crystal structure for a construct lacking the seven C-terminal amino acids. Its quaternary interactions with known protein partners are in good agreement with prior competitive binding assays. This construct can be used for further NMR studies, including protein-protein interaction studies and assessing the impact of environmental conditions on C3 structure and dynamics within C123 as it transitions from monomer to amyloid form.

Characterization of an intermolecular quaternary interaction between discrete segments of the Streptococcus mutans adhesin P1 by 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 polypeptide, 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. However, with sizes of ~ 51 and ~ 185 kDa, respectively, C123 and full-length P1 are too large to achieve high-resolution data for full structural analysis by NMR. Here, we report on 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. Also evaluated are C3's interaction with C12 and the adhesion-inhibiting monoclonal antibody (MAb) 6-8C. NMR titration experiments with 15 N-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-A3VP1 interaction 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. DATABASE: BMRB submission code: 27935.