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 ␤-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.Dental caries (also called tooth decay or dental cavities) is a ubiquitous worldwide disease that affects humans of all age groups. Streptococcus mutans, a primary etiological agent of human dental caries (1) and an increasingly recognized cause of bacterial endocarditis (2), adheres to proteins contained within the salivary pellicle on the tooth surface, the extracellular matrix, and other microbial species (3). Antigen I/II (AgI/II, 2 also known as P1, B, SpaP, or PAc) of S. mutans has been implicated in bacterial adherence to constituents of the salivary pellicle (4, 5) and has been studied for the past three decades as a target for protective immunity against dental caries. Apart from adherence, AgI/II influences biofilm formation (6) and promotes platelet aggregation (7), collagen-dependent bacterial invasion of dentin (8), and cariogenicity (9). Although AgI/II was initially discovered on oral streptococci, it has also been identified in members of the Group A and Group B streptococci (10), suggesting a role for this adhesin in a variety of species.The AgI/II family proteins range from 140 to 180 kDa in predicted size and have a primary sequence composed of multiple conserved regions (Fig. 1b). Toward the N terminus, repeated sequences of high alanine content constitute the alanine-rich region followed by a segment commonly referred to as the variable (V) region. Further C-terminal in the sequence is a region of high proline content that forms a repetitive prolinerich region. Following the AgI/II proline-rich region is a C-terminal region (60 kDa or 550 amino acids), which is the most conserved region of AgI/II, with 62% identity ...

Structural and Functional Analysis of Cell Wall-anchored Polypeptide Adhesin BspA in Streptococcus agalactiae

“…6A and comprise a pair of compact subdomains (residues 554 -725 and 726 -881 of full-length BspA) fused by a linker. The two subdomains are equivalent to those termed C2 and C3 in other AgI/II family polypeptides (13)(14)(15)(16). The absence of a C1 subdomain, a feature that is present in all other AgI/II family proteins characterized to date, accounts for the size disparity between BspA-C domain and other AgI/II family C domains.…”

“…Initially, we focused our efforts on the BspA C-terminal domain (BspA-C, 328-aa residues). Although our whole cell binding studies identify the BspA variable domain as being responsible for gp340 binding by this polypeptide, a role for the C-terminal domain of AgI/II family polypeptides in target binding has been suggested by others (9,15,39). Despite being significantly shorter (328-aa residues as compared with 502-508 aa in other AgI/II family proteins), the BspA-C domain exhibits a high degree of sequence identity to equivalent regions in other AgI/II family proteins, indicative of analogous function.…”

“…Crystal structures of the V regions of SpaP and SspB have revealed a common architecture consisting of a lectin-like fold with a putative binding cleft (11,12). The structure of the C-terminal subdomains C1, C2, and C3 has also been elucidated by x-ray crystallography and found to consist of ␤-sandwich domains stabilized by isopeptide bonds (13)(14)(15)(16).…”

“…The absence of a C1 subdomain, a feature that is present in all other AgI/II family proteins characterized to date, accounts for the size disparity between BspA-C domain and other AgI/II family C domains. The C1-C2 interface region has been suggested as having a role in carbohydrate binding in the S. mutans AgI/II family protein SpaP (15). Its absence in BspA, and by inference from sequence analysis of BspB-D (Fig.…”

“…Previously reported crystal structures of AgI/II family polypeptide C-terminal domains have identified the presence of bound metal ions, which have been suggested to play a role in enhancing stability and conferring adhesive properties (13)(14)(15)(16). By contrast, no bound metal ions are observed in the crystal structures of BspA-C G744D or BspA-C. BspA-C2 subdomain does possess a putative unoccupied metal-binding site, equivalently positioned to those reported in SpaP-C2, SspB-C2, and AspA-C2.…”