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

Dolleweerd, Craig J. van; Kelly, Charles; Chargelegue, Daniel; K.‐C., Julian

doi:10.1074/jbc.m400820200

Cited by 19 publications

(24 citation statements)

References 39 publications

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“…Corresponding well with the current A 3 VP 1 crystal structure, a single A repeat adopting an α-helix was proposed to match in length a single P repeat adopting a PPII helix (17). Alanine-and proline-rich repeats are highly conserved among AgI/II family members, although the total number of repeats varies between one and five ( Fig.…”

Section: Discussionsupporting

confidence: 72%

“…In addition to the anti-AgI/II MAbs described in this study, antibodies that inhibit S. mutans adherence to SAG have also been mapped to the A region (17,(34)(35)(36)(37). Notably, two-thirds of the surface area of the A/P stalk of A 3 VP 1 is formed by the A 3 sequence.…”

Section: Discussionmentioning

confidence: 97%

“…Studies aimed at developing active and passive immune therapies have analyzed segments of Ag I/II that contribute to bacterial adherence and cariogenicity (2, 16). Several anti-AgI/II monoclonal antibodies (MAbs) recognize complex conformational epitopes encompassing noncontiguous sequences within the A and the P repeats (16,17), indicating that these regions are in close proximity, but the exact nature of the intramolecular interaction required to achieve a functional adhesin was undetermined.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 structure (155 Å) formed by the intimate association of two widely separated segments within the primary sequence. The A 3 repeat of the alanine-rich domain was found to adopt a long α-helical structure that intertwines with the P 1 repeat polyproline type II (PPII) helix to form a highly extended stalk.…”

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Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of α- and PPII-helices

Larson

Rajashankar

Patel

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

175

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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...

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Section: Discussionsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 97%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of α- and PPII-helices

Larson

Rajashankar

Patel

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

175

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“…One of these, a synthetic 20-aa-residue peptide designated p1025, comprising 1,025 to 1,044 aa of AgI/II from S. mutans, blocks the binding of streptococci to gp340 (292). Epitopes within the A and P regions are in close proximity in native AgI/II (622), and so it is believed that the A and P regions interact to orientate the V region to bind oligosaccharides. Ag I/II-binding domains for P. gingivalis are discussed below (see Community Development).…”

Section: Cell Wall-anchored Polypeptidesmentioning

confidence: 99%

StreptococcusAdherence and Colonization

Nobbs

Lamont

Jenkinson

2009

Microbiol Mol Biol Rev

552

569

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SUMMARY Streptococci readily colonize mucosal tissues in the nasopharynx; the respiratory, gastrointestinal, and genitourinary tracts; and the skin. Each ecological niche presents a series of challenges to successful colonization with which streptococci have to contend. Some species exist in equilibrium with their host, neither stimulating nor submitting to immune defenses mounted against them. Most are either opportunistic or true pathogens responsible for diseases such as pharyngitis, tooth decay, necrotizing fasciitis, infective endocarditis, and meningitis. Part of the success of streptococci as colonizers is attributable to the spectrum of proteins expressed on their surfaces. Adhesins enable interactions with salivary, serum, and extracellular matrix components; host cells; and other microbes. This is the essential first step to colonization, the development of complex communities, and possible invasion of host tissues. The majority of streptococcal adhesins are anchored to the cell wall via a C-terminal LPxTz motif. Other proteins may be surface anchored through N-terminal lipid modifications, while the mechanism of cell wall associations for others remains unclear. Collectively, these surface-bound proteins provide Streptococcus species with a “coat of many colors,” enabling multiple intimate contacts and interplays between the bacterial cell and the host. In vitro and in vivo studies have demonstrated direct roles for many streptococcal adhesins as colonization or virulence factors, making them attractive targets for therapeutic and preventive strategies against streptococcal infections. There is, therefore, much focus on applying increasingly advanced molecular techniques to determine the precise structures and functions of these proteins, and their regulatory pathways, so that more targeted approaches can be developed.

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Using peptides to study the interaction between the p53 tetramerization domain and HIV‐1 tat

et al. 2008

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Peptides are valuable tools for studying protein-protein interactions, especially in cases of isolated protein domains and natively unfolded proteins. Here, we used peptides to quantitatively characterize the interaction between the natively unfolded HIV-1 Tat protein and the tetramerization domain of the cellular tumor suppressor protein p53. We used peptide mapping, fluorescence anisotropy, and NMR spectroscopy to perform a detailed structural and biophysical characterization of the interaction between the two proteins and elucidate its molecular mechanism, which have so far been studied using cell-based methods. We show that the p53 tetramerization domain, p53(326-355), binds directly to residues 1-35 and 47-57 in Tat. We have characterized the interaction between p53(326-355) and Tat(47-57) in detail. The p53 residues that are mainly involved in binding to Tat(47-57) are E343 and E349, which bind to the positively charged arginine-rich motif of Tat by a partly electrostatic mechanism. All oligomerization states of p53(326-355) bind Tat(47-57) without inhibiting p53 tetramerization, since the residues in p53(326-355) that bind Tat(47-57) face away from the tetramerization interface. We conclude that p53 is able to bind Tat as a transcriptionally active tetramer.

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

Cited by 19 publications

References 39 publications

Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of α- and PPII-helices

Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of α- and PPII-helices

StreptococcusAdherence and Colonization

Using peptides to study the interaction between the p53 tetramerization domain and HIV‐1 tat

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