The studies in this dissertation defined the molecular mechanism of prothrombin (ProT) activation by a recently-identified Staphylococcus aureus exoprotein, von Willebrand factor-binding protein (VWbp). We demonstrated that VWbp activates ProT conformationally in a mechanism requiring insertion of its NH2-terminal Val1-Val2 residues into the NH2-terminal binding cleft on ProT. Such non-proteolytic activation of ProT has been previously identified for only one other protein, staphylocoagulase (SC), which shares structural homology with VWbp. Kinetic studies of ProT activation by VWbp showed that it activates ProT by a unique substrate-dependent, hysteretic mechanism. The NH2-terminal domains of VWbp (VWbp(1-263)) bind weakly to ProT, forming an inactive complex that is fully activated through a slow conformational change mediated by binding of tripeptide substrates or the physiological substrate, fibrinogen (Fbg). The activity of VWbp(1-474) is increased compared to VWbp(1-263), supporting a contribution from the COOH-terminal region in ProT activation. The role of allostery in regulating binding and activation of ProT by VWbp was also demonstrated from the effect of the fragment 1 and 2 domains of ProT on VWbp function, as well as the cofactor effect of substrate on complex formation. Specifically, loss of fragment 1 of ProT or active-site occupation enhances affinity for both VWbp(1-263) and VWbp(1-474), suggesting a regulatory role for fragment 1 and verifying the substrate dependence of the activating conformational change. Further, the activation state of anion-binding (pro)exosite I influences ProT-VWbp binding. This pattern is consistent with that seen during the physiological pathway of ProT activation, where high-affinity exosite expression is linked to formation of a competent active site. Slow, progressive loss of the fragment domains of ProT*VWbp occurs through autocatalysis, reinforcing the potential role of these domains in ProT activation during S. aureus infections. Together, these findings reveal a pattern of regulation of ligand recognition similar to that of normal thrombin, but with strictly defined recognition of Fbg by the ProT*VWbp complex. The restricted substrate specificity provided by the hysteretic activation mechanism may allow VWbp to serve as a virulence factor in propagating the growth of fibrin-rich vegetations during acute infective endocarditis.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-12162009-101657 |
| Date | 04 January 2010 |
| Creators | Kroh, Heather K. |
| Contributors | Paul E. Bock, Richard L. Hoover |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-12162009-101657/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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