We report here the crystal structure of the minimal ligand-binding segment of the Staphylococcus aureus MSCRAMM, clumping factor A. This ®brinogen-binding segment contains two similarly folded domains. The fold observed is a new variant of the immunoglobulin motif that we have called DE-variant or the DEv-IgG fold. This subgroup includes the ligand-binding domain of the collagen-binding S.aureus MSCRAMM CNA, and many other structures previously classi®ed as jelly rolls. Structure predictions suggest that the four ®brinogen-binding S.aureus MSCRAMMs identi®ed so far would also contain the same DEv-IgG fold. A systematic docking search using the C-terminal region of the ®brinogen g-chain as a probe suggested that a hydrophobic pocket formed between the two DEv-IgG domains of the clumping factor as the ligand-binding site. Mutagenic substitution of residues Tyr256, Pro336, Tyr338 and Lys389 in the clumping factor, which are proposed to contact the terminal residues 408 AGDV 411 of the g-chain, resulted in proteins with no or markedly reduced af®nity for ®brinogen.

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Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 Å) crystal structure of the A 3 VP 1 fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 Å) through the interaction of two noncontiguous regions in the primary sequence. The A 3 repeat of the alanine-rich domain adopts an extended α-helix that intertwines with the P 1 repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that fulllength AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A 3 and P 1 helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine-and proline-rich repeats determining their length.bacterial adhesion | dental caries | Streptococcus | x-ray crystallography | fibrous proteins S treptococcus mutans is the causative agent of human dental caries (1) and its protein adhesin antigen I/II (AgI/II) is a known target of protective immunity (2). AgI/II family molecules are expressed by numerous oral streptococci (3) and homologs have also been identified in the invasive pathogens Streptococcus pyogenes and Streptococcus agalactiae (4) (Fig. S1). In addition to mediating adhesion to the tooth surface (5), AgI/II influences biofilm formation (6), promotes collagen-dependent bacterial invasion of dentin (7), and mediates adherence to human epithelial cells (8). Elimination of AgI/II results in decreased virulence (9), but despite three decades of study, a mechanistic understanding of the functional properties of the molecule has been stymied by a lack of understanding of its structure.Originally identified as AgI/II (10) (also called P1, PAc, or SpaP), members of this protein family contain between 1310 and 1653 amino acids (aa) beginning with an amino-terminal signal motif that directs secretion, followed by the A, V, and P regions (Fig. 1A). The A region typically consists of 3-4 alanine-rich repeats (82 residues each) with 23-30% alanine content. The P region has 3-4 proline-rich repeats (39 residues each) with ∼35% proline content. Nested between the A and P repeats is a segment commonly referred to as the V or variable region, which contains within it a stretch of ∼100 amino acids where most of the sequence variation among S. mutans AgI/II molecules is clustered (11). The crystal structure of the V region adopts a globular β-s...

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 ...

The B region of the Cna collagen adhesin has a novel fold that is reminiscent of but is inverse in nature to the IgG fold. This B region assembly could effectively provide the needed flexibility and stability for presenting the ligand binding A region away from the bacterial cell surface.