Structural and functional variation within the alanine-rich repetitive domain of streptococcal antigen I/II.
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 alpha-helical and may form coiled-coil structures. However, the A region of SspB underwent a conformational change and exhibited reduced alpha-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 alpha-helical structure more rapidly than that of SspA or SpaP when the temperature was increased from 10 to 40 degrees C. Furthermore, the SspB A region polypeptide denatured completely at a temperature that was 7 to 9 degrees 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.
Demuth
Donald R
DR
Department of Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania 19104-6002, USA. demuth@biochem.dental.upenn.edu
Irvine
Douglas C
DC
D018829:Adhesins, Bacterial / Q000737:chemistry / Q000502:physiology; D000409:Alanine; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000502:physiology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D013291:Streptococcus / Q000276:immunology*; D013696:Temperature
D018829:Adhesins, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; C055764:S-layer proteins; C082314:SspA protein, bacteria; D000409:Alanine