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 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.

FIG. 1.

Expression and cleavage of glutathione S-transferase-antigen I/II fusion proteins. DNA fragments encoding the N-terminal repetitive domains of SspA, SspB, and SpaP were cloned into pGEX-6P2 and expressed in E. coli. The resulting fusion proteins were purified on glutathione-Sepharose and cleaved with PreScission protease as described in Materials and Methods. The resulting antigen I/II peptides were purified by FPLC on Sephacryl G-75 and evaluated on SDS-10% PAGE gels stained with Coomassie blue. Lanes: 1, SspB/glutathione S-transferase fusion protein; 2, AR-B peptide; 3, AR-P peptide; 4, SspA/glutathione S-transferase fusion protein; 5, AR-A peptide. Positions of molecular size markers are indicated on the left.

FIG. 2.

Competitive inhibition of salivary gp340-mediated streptococcal aggregation by antigen I/II peptides AR-A (A), AR-B (B), and AR-P (C). The AR-A and AR-B peptides were incubated at 37°C with S. gordonii M5 cells in the presence of 30 μg of gp340 per ml in a 1-ml volume. The AR-P peptide was similarly incubated with S. mutans KPSK2 (Fig. 2C). gp340-mediated bacterial aggregation was followed by monitoring the decrease in OD₆₇₅ as cell aggregates formed and settled. Incubations were carried out in the presence of 10 (♦), 25 (▴), 50 (▪), or 90 μg (▾) of each peptide per ml or in the presence of 30 μg of full-length SspB per ml (•). Positive control reactions (○) consisted of bacteria and gp340 in the absence of AR peptide. Nonspecific settling of cells was monitored in blank reactions containing only bacteria and was ≤0.05 OD₆₇₅ units for all reactions. All reactions were carried out in triplicate.

FIG. 3.

Circular dichroism spectrograph of the antigen I/II peptide AR-B. Circular dichroism spectra of AR-B (100 μg/ml) were recorded from 300 nm to 200 nm on an Aviv 60DS circular dichroism spectrophotometer at 37°C with a scan rate of 1 nm/s. The contents of α-helix, β-sheet, and random coil were deduced from the spectrograph data with the K2d program (1).

FIG. 4.

Effects of temperature (A to C) and pH (D to F) on the stability of AR peptides. To compare the stability of the AR-A (green), AR-B (black), and AR-P (red) peptides as a function of temperature, the ellipticity at 222 nm was determined from 5°C to 80°C in buffers of pH 5.5 (A), pH 7.5 (B), and pH 8.5 (C). Data were acquired with a temperature interval of 1°C. The slope for each plot was determined by linear regression of a data set encompassing the temperature points from 15°C to 37°C for each peptide. The calculated slopes are shown in panels A to C. A positive slope represents an increase in ellipticity at 222 nm, indicating a decrease in the α-helix content. To more clearly represent the influence of pH on the stability of the peptides, the melt curves for AR-A (D), AR-B (E), and AR-P (F) in each buffer were plotted separately. In each panel, the solid lines represent pH 5.5, the dashed lines represent pH 7.5, and the dotted lines represent pH 8.5.

FIG. 5.

(A) Elution profile of standard proteins and AR-B peptide from Superdex 75. Purified AR-B was chromatographed on Superdex 75 (1.6 by 40 cm) as described in Materials and Methods and compared to the elution of the standard proteins bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa), and aprotinin (6.4 kDa). (B) Native gel electrophoresis of S. gordonii SspB protein. Purified SspB protein (15 μg) was electrophoresed in a 4 to 15% gradient PhastGel with native gel buffer strips. Native size standards were thyroglobulin (660 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and bovine serum albumin (66 kDa).