Crystal structure of the C-terminal region of Streptococcus mutans antigen I/II and characterization of salivary agglutinin adherence domains

Abstract

The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 Å resolution crystal structure of the complete C-terminal region of AgI/II. The C-terminal region is comprised of three major domains: C(1), C(2), and C(3). Each domain adopts a DE-variant IgG fold, with two β-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimal region of binding was contained within the first and second DE-variant-IgG domains (C(1) and C(2)) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C(1) and C(2) domains. Competition experiments demonstrated that the C- and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C(1) and C(2) domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.

FIGURE 1.

C-terminal structure and domain layout of AgI/II of S. mutans. a, the AgI/II C-terminal region is composed of three domains, labeled as C₁, C₂, and C₃. The AgI/II C-terminal domains are shown in a ribbon model overlaid with a surface plot to illustrate the overall contiguous structure of the three domains. Each domain has an isopeptide bond linking two β-sheets (shown in red). The C₁ and C₂ domains have five bound glucoses (labeled glc1–5). Additionally, three calcium ions exist in the C₂ and C₃ domains (green), and one magnesium ion exists in the C₁ domain (purple). b, the graphic represents the domain structure of AgI/II, where the alanine-rich and proline-rich repeats, as well as the variable region and C terminus (C₁, C₂, C₃), are indicated with distinct colors.

FIGURE 2.

C-terminal domains of AgI/II. a, each of the AgI/II C-terminal domains is shown using a rainbow-colored ribbon model, where each strand is highlighted from A–G strands as seen in a typical IgG like-fold. Variations in the form of additional strands and helices occur between the D and E strands of this fold and are highlighted in light pink. Isopeptide bonds are indicated with an asterisk. b, superposition of the C₁, C₂, and C₃ domains shows the similarities in these domains.

FIGURE 3.

Interaction of carbohydrates within the C₁₂ cleft. a, the stereo image shows the three glucose molecules, which are bound within the cleft formed between the C₁ and C₂ domains of AgI/II. Sites Glc1 and Glc3 are present in both molecule A and molecule B, whereas Glc2 is only present in molecule A of the asymmetric unit. The DH2-helix of C₂ domain is in close proximity to the cleft, and residues from DH2 interact with Glc1. Each glucose forms bonds with surrounding residues and waters. Glc1 and Glc2 are bridged through a shared water molecule (W). b–d, the interactions between Glc1 (b), Glc2 (c), and Glc3 (d) and AgI/II residues are shown below in a Ligplot diagram (39). e, the electron density map shown in stereo for the Glc2 sugar, which is stabilized in the cleft by hydrogen bonding with nearby residues including the backbone oxygen of isoleucine 1157. The backbone oxygen of the neighboring tyrosine 1156 forms part of a coordination site for the nearby calcium ion.

FIGURE 4.

Comparison of AgI/II C₁₂₃ and SspB C₂₃ structures. a, superpositions of the S. mutans AgI/II (red) and S. gordonii SspB (PDB: 2WOY) (blue) structures are shown as traces of the backbone atoms. The two different structures of the AgI/II family displayed a high degree of similarity, superposing with an average r.m.s.d. of 0.906 Å. The isopeptide bonds (black) of the C₂ and C₃ domains are located at equivalent positions on both structures. The C₂ DH2-helix is indicated with an asterisk. b, a stereo view of the superposition of the DH2-helices from both AgI/II and SspB structures, highlighting the mutations that occur in this region.

FIGURE 5.

Evaluation of AgI/II polypeptide-SAG interactions using surface plasmon resonance. a, SAG was immobilized, and AgI/II polypeptides (2 μm each) were injected over the CM5 chip surface. Recombinant full-length AgI/II (CG14, black), C₁₂₃ (orange), and C₁₂ (red) each displayed a strong binding response (RU, resonance units; 1 resonance unit = ∼1 pg/mm²) accompanied by slower dissociations that are indicative of adherence to the SAG-coated surface. b, the interaction between C₁₂ with immobilized SAG at multiple concentrations is shown. Kinetics fitting with multiple C₁₂ concentrations estimated the K_D to be 57 nm. c, competition experiments were performed for the binding of AVP, A₃VP₁, or C₁₂₃ to immobilized SAG. The adherence of AgI/II fragments to SAG without competition (labeled as None) is shown first followed by measured adherence when competed against other AgI/II fragments (labeled CG14, C₁₂₃, AVP, or A₃VP₁). All experiments were carried out in triplicate. Error bars indicate mean ± S.D.

FIGURE 6.

Electron micrographs and composite model of AgI/II of S. mutans. a, electron microscopy of native AgI/II obtained from streptococcal cells (S. mutans Guy's strain) is shown with a composite of electron micrographs of unidirectionally metal-shadowed streptococcal AgI/II molecules at magnification ×235,000 (bar = 50 nm). The micrographs of AgI/II illustrate an elongated model with globular domains present at the termini. The longer globular C terminus (∼13 nm) present at an opposing end from the smaller V-region (∼6 nm) can be clearly visualized in these micrographs. The end-to-end length of the molecules is 65.8 (S.D. ± 3.6) nm, similar to predicted dimensions from analytical ultracentrifugation of AgI/II (16). Southern blotting (40) and partial DNA sequencing of AgI/II (41) have displayed a high degree of homology (>95%) between S. mutans NG8 and Guy's strains. b, illustrated is a composite model of tertiary structure for AgI/II, including the extended hybrid of the α-helical A-region (red) and the polyproline type II helical P-region (blue), that separates the globular V-region(green) and C-terminal region (light blue, C₁; yellow, C₂; blue, C₃). The two distinct and non-competing binding sites for SAG are contained within the A₃VP₁ and C₁₂ regions, which are located at opposite ends of the A/P stalk. Following the C₃ domain is the LPXTG motif that anchors the protein to the cell wall peptidoglycan layer.