Abstract
Many bacterial pathogens secrete proteins that activate host trypsinogen-like enzyme precursors, most notably the proenzymes of the blood coagulation and fibrinolysis systems1,2. Staphylococcus aureus, an important human pathogen implicated in sepsis and endocarditis3, secretes the cofactor staphylocoagulase, which activates prothrombin, without the usual proteolytic cleavages, to directly initiate blood clotting4,5. Here we present the 2.2 Å crystal structures of human α-thrombin and prethrombin-2 bound to a fully active staphylocoagulase variant. The cofactor consists of two domains, each with three-helix bundles; this is a novel fold that is distinct from known serine proteinase activators, particularly the streptococcal plasminogen activator streptokinase6. The staphylocoagulase fold is conserved in other bacterial plasma-protein-binding factors and extracellular-matrix-binding factors7,8,9. Kinetic studies confirm the importance of isoleucine 1 and valine 2 at the amino terminus of staphylocoagulase for zymogen activation. In addition to making contacts with the 148 loop and (pro)exosite I of prethrombin-2, staphylocoagulase inserts its N-terminal peptide into the activation pocket of bound prethrombin-2, allosterically inducing functional catalytic machinery. These investigations demonstrate unambiguously the validity of the zymogen-activation mechanism known as ‘molecular sexuality’10.
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References
Boyle, M. D. & Lottenberg, R. Plasminogen activation by invasive human pathogens. Thromb. Haemost. 77, 1–10 (1997)
Lähteenmäki, K., Kuusela, P. & Korhonen, T. K. Bacterial plasminogen activators and receptors. FEMS Microbiol. Rev. 25, 531–552 (2001)
Mylonakis, E. & Calderwood, S. B. Medical progress: Infective endocarditis in adults. N. Engl. J. Med. 345, 1318–1330 (2001)
Hemker, H. C., Bas, B. M. & Muller, A. D. Activation of a pro-enzyme by a stoichiometric reaction with another protein. The reaction between prothrombin and staphylocoagulase. Biochim. Biophys. Acta 379, 180–188 (1975)
Kawabata, S. et al. Structure and function relationship of staphylocoagulase. J. Prot. Chem. 6, 17–32 (1987)
Wang, X., Lin, X., Loy, J. A., Tang, J. & Zhang, X. C. Crystal structure of the catalytic domain of human plasmin complexed with streptokinase. Science 281, 1662–1665 (1998)
Bjerketorp, J. et al. A novel von Willebrand factor binding protein expressed by Staphylococcus aureus. Microbiology 148, 2037–2044 (2002)
Tettelin, H. et al. Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae. Proc. Natl Acad. Sci. USA 99, 12391–12396 (2002)
Glaser, P. et al. Genome sequence of Streptococcus agalactiae, a pathogen causing invasive neonatal disease. Mol. Microbiol. 45, 1499–1513 (2002)
Bode, W. & Huber, R. Induction of the bovine trypsinogen–trypsin transition by peptides sequentially similar to the N-terminus of trypsin. FEBS Lett. 68, 231–236 (1976)
Huber, R. & Bode, W. Structural basis of the activation and action of trypsin. Acc. Chem. Res. 11, 114–122 (1978)
Khan, A. R. & James, M. N. Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes. Protein Sci. 7, 815–836 (1998)
Boxrud, P. D., Verhamme, I. M., Fay, W. P. & Bock, P. E. Streptokinase triggers conformational activation of plasminogen through specific interactions of the amino-terminal sequence and stabilizes the active zymogen conformation. J. Biol. Chem. 276, 26084–26089 (2001)
Wang, S., Reed, G. L. & Hedstrom, L. Deletion of Ile1 changes the mechanism of streptokinase: evidence for the molecular sexuality hypothesis. Biochemistry 38, 5232–5240 (1999)
Vijayalakshmi, J., Padmanabhan, K. P., Mann, K. G. & Tulinsky, A. The isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin: changes accompanying activation and exosite binding to thrombin. Protein Sci. 3, 2254–2271 (1994)
Bode, W. et al. The refined 1.9 Å crystal structure of human α-thrombin: interaction with d-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 8, 3467–3475 (1989)
Parry, M. A. et al. The ternary microplasmin–staphylokinase–microplasmin complex is a proteinase–cofactor–substrate complex in action. Nature Struct. Biol. 5, 917–923 (1998)
Anderson, P. J., Nesset, A., Dharmawardana, K. R. & Bock, P. E. Characterization of proexosite I on prothrombin. J. Biol. Chem. 275, 16428–16434 (2000)
Bock, P. E. Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones II. Properties of thrombin derivatives as reporters of prothrombin fragment 2 binding and specificity of the labeling approach for other proteinases. J. Biol. Chem. 267, 14974–14981 (1992)
Tager, M. & Drummond, M. C. Staphylocoagulase. Ann. NY Acad. Sci. 128, 92–111 (1965)
Bock, P. E., Olson, S. T. & Björk, I. Inactivation of thrombin by antithrombin is accompanied by inactivation of regulatory exosite I. J. Biol. Chem. 272, 19837–19845 (1997)
Cheung, A. I., Projan, S. J., Edelstein, R. E. & Fischetti, V. A. Cloning, expression, and nucleotide sequence of a Staphylococcus aureus gene (fbpA) encoding a fibrinogen-binding protein. Infect. Immun. 63, 1914–1920 (1995)
Jeng, A. et al. Molecular genetic analysis of a group A Streptococcus operon encoding serum opacity factor and a novel fibronectin-binding protein, SfbX. J. Bacteriol. 185, 1208–1217 (2003)
Joh, D., Wann, E. R., Kreikemeyer, B., Speziale, P. & Höök, M. Role of fibronectin-binding MSCRAMMs in bacterial adherence and entry into mammalian cells. Matrix Biol. 18, 211–223 (1999)
Schwarz-Linek, U. et al. Pathogenic bacteria attach to human fibronectin through a tandem β-zipper. Nature 423, 177–181 (2003)
Acknowledgements
We are grateful to Diagnostica Stago for the S. aureus strain, to R. Mentele for sequencing, to G. Bourenkov for help during data collection, and to S. Iwanaga for initiating the project. We thank E. Consacro, H. Kroh and S. Stuart for excellent technical assistance. This work was supported by a National Institutes of Health (NIH) grant to P.E.B., as well as funding from SFB469, the EU project SPINE and the Fonds der Chemischen Industrie. P.P. and P.J.A were supported in part by NIH training grants.
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Supplementary information
41586_2003_BFnature01962_MOESM1_ESM.jpg
Supplementary Figure: The sequence of the crystallized SC (S. aureus Newman 2D, strain Tager 104) is compared with two other SCs and several homologs. Strictly conserved residues and conservative substitutions throughout are highlighted in red and pink, respectively; and additional conserved residues are highlighted in yellow. SC residues that contact the 148-loop or exosite I are indicated with closed or open triangles, respectively. The conserved RGD motif in SfbX is shadowed blue. (JPG 281 kb)
41586_2003_BFnature01962_MOESM2_ESM.jpg
Supplementary Figure: Stereo ribbon plot showing the symmetric dimer present in the asymmetric unit, with thrombin and SC moieties colored yellow and orange, and red and salmon, respectively. The terminal SC residues as well as single helices are labeled. (JPG 150 kb)
41586_2003_BFnature01962_MOESM3_ESM.ppt
Supplementary Figure: A superimposition of the µ-plasmin·SK (yellow) and Pre-2·SC(325) (red) complexes. Note the different arrangement around the respective proteinase. (PPT 549 kb)
41586_2003_BFnature01962_MOESM4_ESM.mov
Supplementary Movie: Zoom from standard orientation into the activation pocket of Pre-2. SC is coloured yellow, the surface of Pre-2 according to the surface potential (blue: positive, red: negative). (MOV 982 kb)
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Friedrich, R., Panizzi, P., Fuentes-Prior, P. et al. Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation. Nature 425, 535–539 (2003). https://doi.org/10.1038/nature01962
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DOI: https://doi.org/10.1038/nature01962
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