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Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin*

  • Author Footnotes
    1 These authors contributed equally to this work.
    Barbara A. Bensing
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Division of Infectious Diseases, Veterans Affairs Medical Center, Department of Medicine, University of California, San Francisco and the Northern California Institute for Research and Education, San Francisco, California 94121
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Lioudmila V. Loukachevitch
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Kathryn M. McCulloch
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232
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  • Hai Yu
    Affiliations
    Department of Chemistry, University of California, Davis, California 95616
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  • Kendra R. Vann
    Affiliations
    Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232
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  • Zdzislaw Wawrzak
    Affiliations
    Life Sciences Collaborative Access Team, Synchrotron Research Center, Northwestern University, Argonne, Illinois 60439
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  • Spencer Anderson
    Affiliations
    Life Sciences Collaborative Access Team, Synchrotron Research Center, Northwestern University, Argonne, Illinois 60439
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  • Xi Chen
    Affiliations
    Department of Chemistry, University of California, Davis, California 95616
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  • Paul M. Sullam
    Affiliations
    Division of Infectious Diseases, Veterans Affairs Medical Center, Department of Medicine, University of California, San Francisco and the Northern California Institute for Research and Education, San Francisco, California 94121
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  • T.M. Iverson
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232

    Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232

    Department of Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232

    Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232
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  • Author Footnotes
    * This work was supported by the Department of Veterans Affairs, National Institutes of Health Grants AI41513 (to P. M. S.) and AI106987 (to T. M. I. and P. M. S.), American Heart Association Grant 14GRNT20390021 (to T. M. I.), National Institutes of Health Training Grant in Molecular Biophysics T32 GM008320 and Education Grant for the Initiative to Maximize Student Development R25 GM062459 (to K. R. V.), and National Institutes of Health Training Grant in Mechanisms of Vascular Disease T32 HL007751 (to K. M. M.). A portion of the experiments described here used the Vanderbilt PacVan robotic crystallization facility, which was supported by National Institutes of Health Grant S10 RR026915. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    ♦ This article was selected as a Paper of the Week.
    1 These authors contributed equally to this work.
Open AccessPublished:February 01, 2016DOI:https://doi.org/10.1074/jbc.M115.701425
      Streptococcus sanguinis is a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets. S. sanguinis expresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site.

      Introduction

      Infective endocarditis is associated with significant morbidity and mortality (
      • Hoen B.
      • Duval X.
      Clinical practice. Infective endocarditis.
      ). Although adherence between pathogen and host platelets is one of the first required steps for infection (
      • Fitzgerald J.R.
      • Foster T.J.
      • Cox D.
      The interaction of bacterial pathogens with platelets.
      ,
      • Sullam P.M.
      • Sande M.A.
      Role of platelets in endocarditis: clues from von Willebrand disease.
      ), the pathogenesis of infective endocarditis is a complex process that is not well understood.
      The viridans group of streptococci, including Streptococcus sanguinis and Streptococcus gordonii, accounts for an estimated 17–45% of all cases of infective endocarditis (
      • Murdoch D.R.
      • Corey G.R.
      • Hoen B.
      • Miró J.M.
      • Fowler Jr., V.G.
      • Bayer A.S.
      • Karchmer A.W.
      • Olaison L.
      • Pappas P.A.
      • Moreillon P.
      • Chambers S.T.
      • Chu V.H.
      • Falcó V.
      • Holland D.J.
      • Jones P.
      • et al.
      Clinical presentation, etiology, and outcome of infective endocarditis in the 21st century: the International Collaboration on Endocarditis-Prospective Cohort Study.
      ,
      • Thornhill M.H.
      • Dayer M.J.
      • Forde J.M.
      • Corey G.R.
      • Chu V.H.
      • Couper D.J.
      • Lockhart P.B.
      Impact of the NICE guideline recommending cessation of antibiotic prophylaxis for prevention of infective endocarditis: before and after study.
      ,
      • Tleyjeh I.M.
      • Steckelberg J.M.
      • Murad H.S.
      • Anavekar N.S.
      • Ghomrawi H.M.
      • Mirzoyev Z.
      • Moustafa S.E.
      • Hoskin T.L.
      • Mandrekar J.N.
      • Wilson W.R.
      • Baddour L.M.
      Temporal trends in infective endocarditis: a population-based study in Olmsted County, Minnesota.
      ). These infective endocarditis-associated pathogens often contain a gene encoding a serine-rich repeat adhesin that can mediate the attachment of each respective pathogen to human platelet glycans (
      • Bensing B.A.
      • López J.A.
      • Sullam P.M.
      The Streptococcus gordonii surface proteins GspB and Hsa mediate binding to sialylated carbohydrate epitopes on the platelet membrane glycoprotein Ibα.
      ,
      • Plummer C.
      • Wu H.
      • Kerrigan S.W.
      • Meade G.
      • Cox D.
      • Ian Douglas C.W.
      A serine-rich glycoprotein of Streptococcus sanguis mediates adhesion to platelets via GPIb.
      ,
      • Takamatsu D.
      • Bensing B.A.
      • Cheng H.
      • Jarvis G.A.
      • Siboo I.R.
      • Lopez J.A.
      • Griffiss J.M.
      • Sullam P.M.
      Binding of the Streptococcus gordonii surface glycoproteins GspB and Hsa to specific carbohydrate structures on platelet membrane glycoprotein Ibα.
      ). The adhesive interaction is perhaps best understood in S. gordonii strain M99, which interacts with platelets via the serine-rich repeat adhesin GspB. This surface component mediates host adherence via a binding region that promotes a high affinity interaction to a narrow range of sialylated carbohydrates (
      • Deng L.
      • Bensing B.A.
      • Thamadilok S.
      • Yu H.
      • Lau K.
      • Chen X.
      • Ruhl S.
      • Sullam P.M.
      • Varki A.
      Oral streptococci utilize a Siglec-like domain of serine-rich repeat adhesins to preferentially target platelet sialoglycans in human blood.
      ). Studies using recombinant GspB and whole bacteria have shown that the binding of GspB to the sialylated glycans of GPIbα is the primary adhesive interaction to host platelets (
      • Bensing B.A.
      • López J.A.
      • Sullam P.M.
      The Streptococcus gordonii surface proteins GspB and Hsa mediate binding to sialylated carbohydrate epitopes on the platelet membrane glycoprotein Ibα.
      ,
      • Takamatsu D.
      • Bensing B.A.
      • Cheng H.
      • Jarvis G.A.
      • Siboo I.R.
      • Lopez J.A.
      • Griffiss J.M.
      • Sullam P.M.
      Binding of the Streptococcus gordonii surface glycoproteins GspB and Hsa to specific carbohydrate structures on platelet membrane glycoprotein Ibα.
      ,
      • Takamatsu D.
      • Bensing B.A.
      • Prakobphol A.
      • Fisher S.J.
      • Sullam P.M.
      Binding of the streptococcal surface glycoproteins GspB and Hsa to human salivary proteins.
      ) and that expression of GspB enhances virulence (
      • Xiong Y.Q.
      • Bensing B.A.
      • Bayer A.S.
      • Chambers H.F.
      • Sullam P.M.
      Role of the serine-rich surface glycoprotein GspB of Streptococcus gordonii in the pathogenesis of infective endocarditis.
      ).
      S. sanguinis is often cited as the most common cause of bacterial infective endocarditis and contains a sequence homolog of GspB (
      • Plummer C.
      • Wu H.
      • Kerrigan S.W.
      • Meade G.
      • Cox D.
      • Ian Douglas C.W.
      A serine-rich glycoprotein of Streptococcus sanguis mediates adhesion to platelets via GPIb.
      ). In S. sanguinis strain SK36, this homolog is called SrpA and includes a binding region (termed SrpABR) that is 32% identical and 46% similar to the corresponding region of GspB (termed GspBBR), but is significantly shorter, suggesting that GspBBR contains an additional domain as compared with SrpABR. Experimental validation of a role for SrpA in virulence is less clear than it is for GspB. Targeted mutagenesis of SrpA and other possible adhesins of S. sanguinis indicated that the deletion of SrpA does not influence virulence in an animal model of infective endocarditis (
      • Turner L.S.
      • Kanamoto T.
      • Unoki T.
      • Munro C.L.
      • Wu H.
      • Kitten T.
      Comprehensive evaluation of Streptococcus sanguinis cell wall-anchored proteins in early infective endocarditis.
      ). However, the subsequent finding that individual deletion of every identified cell wall anchored protein in S. sanguinis had no significant influence on virulence suggests that S. sanguinis could have multiple adhesins that provide functional redundancy (
      • Turner L.S.
      • Kanamoto T.
      • Unoki T.
      • Munro C.L.
      • Wu H.
      • Kitten T.
      Comprehensive evaluation of Streptococcus sanguinis cell wall-anchored proteins in early infective endocarditis.
      ).
      Like GspB, SrpA binds platelet glycoprotein GPIbα and can mediate binding to platelets in vitro (
      • Plummer C.
      • Wu H.
      • Kerrigan S.W.
      • Meade G.
      • Cox D.
      • Ian Douglas C.W.
      A serine-rich glycoprotein of Streptococcus sanguis mediates adhesion to platelets via GPIb.
      ). Unlike GspB, SrpA appears to have a wider range of glycan-binding partners, but quantification of binding between SrpA and defined sialoglycans suggests only weak interactions for the carbohydrates tested (
      • Deng L.
      • Bensing B.A.
      • Thamadilok S.
      • Yu H.
      • Lau K.
      • Chen X.
      • Ruhl S.
      • Sullam P.M.
      • Varki A.
      Oral streptococci utilize a Siglec-like domain of serine-rich repeat adhesins to preferentially target platelet sialoglycans in human blood.
      ). Sialoglycan array data further indicate that although GspB strongly prefers the N-acetylneuraminic acid (Neu5Ac) form of sialic acid, SrpA has some preference for glycans containing the N-glycolylneuraminic acid (Neu5Gc) form of sialic acid (
      • Deng L.
      • Bensing B.A.
      • Thamadilok S.
      • Yu H.
      • Lau K.
      • Chen X.
      • Ruhl S.
      • Sullam P.M.
      • Varki A.
      Oral streptococci utilize a Siglec-like domain of serine-rich repeat adhesins to preferentially target platelet sialoglycans in human blood.
      ), which is not present in humans. Nevertheless, SrpA binds human platelets with high affinity (
      • Plummer C.
      • Wu H.
      • Kerrigan S.W.
      • Meade G.
      • Cox D.
      • Ian Douglas C.W.
      A serine-rich glycoprotein of Streptococcus sanguis mediates adhesion to platelets via GPIb.
      ); thus it is possible that a high affinity human carbohydrate ligand for SrpA remains unidentified.
      The naturally occurring range of carbohydrate binding properties of GspB and SrpA can offer insights into the adhesive properties important for endovascular infection and may allow broader conclusions to be drawn about carbohydrate binding in these related adhesins. In previous work, we determined the crystal structure of the carbohydrate-binding region of GspB (
      • Pyburn T.M.
      • Bensing B.A.
      • Xiong Y.Q.
      • Melancon B.J.
      • Tomasiak T.M.
      • Ward N.J.
      • Yankovskaya V.
      • Oliver K.M.
      • Cecchini G.
      • Sulikowski G.A.
      • Tyska M.J.
      • Sullam P.M.
      • Iverson T.M.
      A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors.
      ,
      • Pyburn T.M.
      • Yankovskaya V.
      • Bensing B.A.
      • Cecchini G.
      • Sullam P.M.
      • Iverson T.M.
      Purification, crystallization and preliminary x-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB.
      ). This binding region (termed GspBBR) contains three linearly arranged, independently folded domains termed the CnaA, Siglec, and Unique domains based upon their structural similarity to other proteins. The binding pocket for sialylated carbohydrates is located within the Siglec domain. Site-directed mutagenesis of residues in the GspB binding pocket identified residues critical for both carbohydrate binding and platelet binding, as well as for virulence in the setting of infective endocarditis (
      • Pyburn T.M.
      • Bensing B.A.
      • Xiong Y.Q.
      • Melancon B.J.
      • Tomasiak T.M.
      • Ward N.J.
      • Yankovskaya V.
      • Oliver K.M.
      • Cecchini G.
      • Sulikowski G.A.
      • Tyska M.J.
      • Sullam P.M.
      • Iverson T.M.
      A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors.
      ).
      Here, we determine the crystal structure of the SrpA carbohydrate-binding region (SrpABR), both alone and in complex with a Neu5Gc-based synthetic sialyl galactoside disaccharide, and we use mutagenesis to validate key residues important for platelet binding. We identified a Thr-Arg motif conserved across homologous carbohydrate-binding adhesins that may orient the sialic acid moiety of carbohydrate ligands. Together, our results suggest that a small number of short sequence insertions in closely related homologs influence the quaternary structure, the tertiary structure, and the binding pocket. The latter may contribute to the differences in carbohydrate affinity and binding spectrum between SrpA and GspB.

      Author Contributions

      B. A. B. performed binding studies, mutagenesis, and data analysis. L. V. L. crystallized SrpABR, collected diffraction data, and performed dynamic light scattering. K. M. M. performed computational studies, assisted with data analysis and structure refinement, and made the figures. H. Y. synthesized the sialyl galactose disaccharide. K. R. V. designed constructs and developed purification protocols during the early stages of this project. S. A. and Z. W. processed diffraction data, calculated the initial phases, and performed the initial chain trace. X. C. guided the synthesis of sialyl galactose disaccharide. P. M. S. developed the experimental strategy, guided the study, and wrote the manuscript. T. M. I. designed the experimental strategy, guided the study, expressed and purified protein, refined the structures, and wrote the manuscript.

      Acknowledgments

      Use of the Advanced Photon Source, an Office of Science User Facility operated for the United States Department of Energy Office of Science by Argonne National Laboratory, was supported by the United States Department of Energy under Contract DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corp. and the Michigan Technology Tri-Corridor Grant 085P1000817.

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