Department of Biochemistry and Molecular Biology and McKnight Brain Institute, University of Florida, Gainesville, FL, USA.
National High Magnetic Field Laboratory, University of Florida, Gainesville, FL, USA.
FEBS J. 2020 Jun;287(12):2597-2611. doi: 10.1111/febs.15158. Epub 2019 Dec 17.
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 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.
致龋菌变形链球菌表面定位的 P1 黏附素（又名抗原 I/II 或 PAc）介导蔗糖非依赖性黏附于牙齿表面。先前的研究表明，P1 的 C 端片段（C123，AgII）也会作为一个单独的多肽释放出来，有助于细胞黏附，与细胞表面完整的 P1 特异性相互作用，并形成淀粉样纤维。确定 C123 如何在原子水平上特异性地与 P1 相互作用，对于理解变形链球菌相关的毒力特性至关重要。然而，C123 和全长 P1 的大小分别约为 51 和 185 kDa，太大了，无法通过 NMR 获得高分辨率数据，从而对其进行全面结构分析。在这里，我们报告了单个 C3 结构域与 A3VP1 的生物学相关相互作用，A3VP1 是 P1 表面投影的顶端头部的多肽代表。同时评估了 C3 与 C12 和抑制黏附的单克隆抗体（MAb）6-8C 的相互作用。用 N 富集的 C3 进行 NMR 滴定实验证明了其对 A3VP1 的特异性结合。基于解析的 C3 分配，确定了两个结合位点，近端和远端。用 C3/C12 复合物互补的 NMR 滴定 A3VP1 表明，A3VP1 的结合发生在远端 C3 结合位点，而近端位点被 C12 占据。MAb 6-8C 与 C3 的结合界面与远端位点的 A3VP1 重叠。这些结果共同确定了一个特定的 C3-A3VP1 相互作用，为理解 C123 与细菌表面上 P1 的相互作用以及由此相互作用产生的相关生物学过程提供了基础。数据库：BMRB 提交代码：27935。
