Abstract
Adhesin P1 (aka AgI/II) is an extracellular protein regulating adherence and detachment of Streptococcus mutans in the oral cavity and thus plays a pivotal role in biofilm development and maturation. P1's naturally occurring C-terminal truncation product, Antigen II (AgII), adopts both soluble, monomeric and insoluble, amyloidogenic forms during the bacterial life cycle. Monomeric AgII forms important quaternary interactions with P1's A3VP1 segment that is projected from the bacterial cell surface to promote cell adhesion, while the functional amyloid form of AgII promotes detachment of mature biofilms. The heterologous recombinant 51-kD C123 construct, comprising most of AgII, has been characterized by X-ray crystallography and serves as a functional surrogate of AgII in studies of adhesion and biofilm regulation. C123 contains three structurally similar domains, C1, C2, and C3. Using Alphafold prediction and the C123 crystal structure, we identified domain boundaries within C123 to develop more tractable constructs for NMR studies, including quaternary interactions with other proteins. The C2 domain is of particular interest because it contains several unique helices in addition to the β-sheet fold it shares with the C1 and C3 domains. Here we report the backbone NMR resonance assignments for the C2 construct. Secondary structure predictions from NMR assignments are in good agreement with those anticipated by Alphafold and the observed crystal structure, except for some of the helices suggesting they are more dynamic. We then compare C2 chemical shift perturbations caused by quaternary interactions with recombinant A3VP1, as well as by a monoclonal antibody, MAb 6-8C, known to inhibit bacterial adherence and C123 binding to A3VP1. We note the C2 chemical shift perturbations are markedly different from previously observed interactions of C3 with A3VP1 and MAb 6-8C, providing further insight on how the individual domains of C123 may vary in their ability to mediate bacterial adhesion and formation of functional amyloid. The prior NMR assignment and characterization of C3 combined with the NMR assignment and characterization of C2 described here provide a foundation for further NMR studies, including assignment of C23 and C123 constructs, protein-protein interaction studies of C23 and C123, assessing the impact of environmental conditions on structure and dynamics within C123 as it transitions from monomer to amyloid form, and the functional relevance of having three successive domains with similar tertiary folds.