The proposed research project focuses on the physiologically important interaction of thrombin with Na+, which is at the basis of the procoagulant and prothrombotic functions of the enzyme in the blood. The project builds upon developments from previous NIH support and consists of the following specific aims: 1. complete the mapping of the functional epitope for Na+ binding to human thrombin and elucidate the structural components of Na+ specificity and activation;2. identify the molecular basis of the functional mimicry of Na+ activation recently discovered in murine thrombin;and 3. engineer Na+ activation into other proteases. We will use a combination of kinetic, site-directed mutagenesis and X-ray structural studies.
In specific aim 1, we will target by mutagenesis the A chain and the disulfide bonds of thrombin to reveal their role in Na+ binding and allosteric activation. Residues Y225 and Y184a in the Na+ binding site will be subject to extensive mutagenesis to dissect their role in monovalent cation specificity and activation. These studies will broaden our understanding of thrombin interaction with Na+, the molecular basis of its procoagulant and prothrombotic functions and will impact the study of clotting enzymes and enzymes activated by monovalent cations in general.
In specific aim 2, we will solve the X-ray crystal structure of murine thrombin and mutate a number of residues to restore Na+ activation as seen in the human enzyme. Reverse mutations in the human enzyme will be made in parallel to produce molecular mimicry of Na+ activation as seen in murine thrombin. These studies will fill an important gap in our understanding of a key enzyme that has been the subject of detailed genetic investigation in recent years, but for which no structural information is available and little is known about its biochemical properties.
In specific aim 3, we will use three """"""""host"""""""" platforms, i.e., trypsins from rat and Streptomyces griseus and tissue-type plasminogen activator, to introduce Na+ activation as found in human thrombin and factor Xa, and molecular mimicry of Na+ activation as found in murine thrombin. These studies will formulate a general strategy for the introduction of allosteric activation in a protease scaffold, and will reveal how enzyme activity can be enhanced rationally using Na+ binding or its molecular mimicry, thereby benefiting engineering studies of proteases of medical and biotechnological relevance.
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|Gohara, David W; Di Cera, Enrico (2011) Allostery in trypsin-like proteases suggests new therapeutic strategies. Trends Biotechnol 29:577-85|
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|Pozzi, Nicola; Chen, Zhiwei; Zapata, Fatima et al. (2011) Crystal structures of prethrombin-2 reveal alternative conformations under identical solution conditions and the mechanism of zymogen activation. Biochemistry 50:10195-202|
|Berny-Lang, Michelle A; Hurst, Sawan; Tucker, Erik I et al. (2011) Thrombin mutant W215A/E217A treatment improves neurological outcome and reduces cerebral infarct size in a mouse model of ischemic stroke. Stroke 42:1736-41|
|Gandhi, Prafull S; Chen, Zhiwei; Appelbaum, Eric et al. (2011) Structural basis of thrombin-protease-activated receptor interactions. IUBMB Life 63:375-82|
|Flick, Matthew J; Chauhan, Anil K; Frederick, Malinda et al. (2011) The development of inflammatory joint disease is attenuated in mice expressing the anticoagulant prothrombin mutant W215A/E217A. Blood 117:6326-37|
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