Structural and Functional Variation within the Alanine-Rich Repetitive Domain of Streptococcal Antigen I/II

Abstract

Members of the antigen I/II family of cell surface proteins are highly conserved, multifunctional adhesins that mediate interactions of oral streptococci with other oral bacteria, with cell matrix proteins (e.g., type I collagen), and with salivary glycoproteins, e.g., gp340. The interaction of gp340 (formerly designated salivary agglutinin) with Streptococcus mutans requires an alanine-rich repetitive domain (A region) of antigen I/II that is highly conserved in all members of this family of proteins. In this report, we show that the A regions from the two Streptococcus gordonii M5 antigen I/II proteins (SspA and SspB) interact differently with the salivary gp340 glycoprotein and appear to be structurally distinct. Recombinant polypeptides encompassing the A region of SspA or from a highly related S. mutans antigen I/II protein (SpaP) competitively inhibited the interaction of gp340 with intact S. gordonii and S. mutans cells, respectively. In contrast, an A region polypeptide from SspB was inactive, and furthermore, it did not bind to purified gp340 in vitro. Circular dichroism spectra suggested that all three polypeptides were highly ␣-helical and may form coiled-coil structures. However, the A region of SspB underwent a conformational change and exhibited reduced ␣-helical structure at pH 8.5, whereas the A region polypeptides from SspA and SpaP were relatively stable under these conditions. Melt curves also indicated that at physiological pH, the A region of SspB lost ␣-helical structure more rapidly than that of SspA or SpaP when the temperature was increased from 10 to 40°C. Furthermore, the SspB A region polypeptide denatured completely at a temperature that was 7 to 9°C lower than that required for the A region polypeptide of SspA or SpaP. The full-length SspB protein and the three A region peptides migrated in native gel electrophoresis and column chromatography with apparent molecular masses that were approximately 2-to 2.5-fold greater than their predicted molecular masses. However, sedimentation equilibrium ultracentrifugation data showed that the A region peptides sedimented as monomers, suggesting that the peptides may form nonglobular intramolecular coiled-coil structures under the experimental conditions used. Taken together, our results suggest that the A region of SspB is less stable than the corresponding A regions of SspA and SpaP and that this structural difference may explain, at least in part, the functional variation observed in their interactions with salivary gp340.

Molecular Interactions of Surface Protein Peptides ofStreptococcus gordoniiwith Human Salivary Components

“…In addition, salivary agglutinin is known for its S. mutans-agglutinating properties (37), while AgI/II, SspB, and SspA bind to 300-to 400-kDa salivary agglutinin (5,8,9,37). In the present study, the binding receptor of the SspB peptide was demonstrated to be a salivary component with a high-molecular-weight complex.…”

“…The A regions of PAc and PAg are thought to assume an ␣-helical structure (5,32,44). Recently, new experimental evidence has shown that A regions from the binding proteins (SspA and SpaP) of S. gordonii and S. mutans that bind to salivary components are highly stable and ␣-helical (8). However, the A region of SspB is less stable than the corresponding A regions of SspA and SpaP.…”

“…In previous studies, lysine inhibited whole saliva-and salivary agglutinin-mediated aggregation of both S. mutans and S. sanguis (13), as well as the adherence of S. mutans to adhesin-promoting protein-coated hydroxyapatite (20). Several studies have also reported interactions between the sialic acid residues of human salivary agglutinin and SspB, SspA, and PAc (5,8,31,37). Furthermore, Kelly et al noted that E1037, located outside the A region, might be required for an AgI/II interaction with the sialic acid residue of salivary agglutinin and that the residue corresponding to E1037 is lysine in the sialic acid binding of the AgI/II homologue of S. gordonii (19).…”

Peptide Mapping of a Novel Discontinuous Epitope of the Major Surface Adhesin from Streptococcus mutans

“…Thus, we hypothesize that the PP II-like helix of the proline-rich region interacts with the alanine-rich region, which itself is predicted to adopt an ␣-helical structure (5,(33)(34)(35). The interaction of PP II-like helices with ␣-helices has been described before for turkey pancreatic polypeptide, a 36-amino acid hormone (28,36), where residues 2 to 8 form a PP II-like helix that is closely packed against an ␣-helix formed from residues 14 -32.…”

Responses of Cariogenic Streptococci to Environmental Stresses

“…SpaP is a multi-functional adhesin, facilitating binding of the bacteria to components of the enamel pellicle (Curtiss, 1985;Lee et al, 1989), as well as to collagen and other host proteins (Demuth and Irvine, 2002;Love et al, 1997). Mutants lacking SpaP showed an impaired capacity to attach to salivacoated hydroxylapatite.…”