Structural examination of serpin-protein interactions

描述（由申请人提供）：尽管Kunitz、Kazal和Bowman-Birk家族的蛋白丝氨酸蛋白酶抑制剂通过锁-键式相互作用来抑制目标蛋白酶，但现在已经通过x射线晶体学、核磁共振波谱和FRET显示，蛇蛋白采用了一种基于构象变化的显着机制来抑制一些相同的丝氨酸蛋白酶。该机制涉及蛇形蛋白和蛋白酶的变化，蛇形蛋白反应中心环（RCL）插入其自身b-sheet A的中心，70A以上的蛋白酶从最初的对接位置转位到蛇形蛋白的远端，伴随着蛋白酶活性位点的扭曲和到达最终位置时大部分蛋白酶的“破碎”。尽管这些研究提供了蛇蛋白如何抑制丝氨酸蛋白酶的结构解释，但它们并没有解释为什么采用如此复杂、容易出错的机制。一个未经证实的假设是，在蛇形蛋白和蛋白酶中发生的构象变化随后被用于其他下游事件，无论是通过受体（如LRP）的识别和信号传递，还是通过蛇形蛋白-蛋白酶复合物中的裂解使蛋白酶无法复活。我们提出了三个具体的目标，这些目标将共同评估完整的蛋白酶易位，蛋白酶粉碎序列在功能表达中的重要性，这些功能使蛇形蛋白在许多情况下成为首选的蛋白酶抑制剂。特异性目的1将确定蛇形蛋白和蛋白酶与受体LRP结合的结构要求和功能后果。特异性Aim 2将确定蚊子蛇形蛋白AFXa的结构、其对人因子Xa的抑制机制以及AFXa-Xa复合物与LRP结合和信号传导的特性。特异性Aim 3将确定半胱天冬酶、组织蛋白酶和枯草杆菌样蛋白酶在与蛇蛋白形成共价复合物时发生的构象变化。我们将使用结合亲和力的热力学测量和核磁共振光谱、FRET和x射线晶体学的结构方法，我们已经成功地在上一个资助期使用，并在适当的时候，通过与Dudley Strickland博士的合作，将我们的发现与功能结果联系起来。

DESCRIPTION (provided by applicant): Although protein serine proteinase inhibitors of the Kunitz, Kazal, and Bowman-Birk families make do with lock-and-key type interactions to inhibit target proteinases, serpins have now been shown by x-ray crystallography, NMR spectroscopy and FRET to employ a remarkable conformational change-based mechanism to inhibit some of the same serine proteinases. This mechanism involves changes to both the serpin and the proteinase, with insertion of the serpin reactive center loop (RCL) into the center of its own b-sheet A, and translocation of the proteinase over 70A to the distal end of the serpin from its initial docking position, with concomitant distortion of the proteinase active site and a "crushing" of a large portion of the proteinase upon it reaching its final location. Whereas these studies have provided a structural explanation of how serpins inhibit serine proteinases, they have not explained why such a complex, error-prone, mechanism is employed. An unproven assumption is that the conformational changes that occur in both serpin and proteinase are exploited subsequently for other downstream events, whether recognition and signaling via a receptor such as LRP, or else rendering the proteinase incapable of revival, through cleavage while in the serpin-proteinase complex. We propose three specific aims that together will enable evaluation of the importance of the full proteinase-translocating, proteinase-crushing sequence in expression of the functions that make serpins the preferred class of proteinase inhibitors under many circumstances. Specific Aim 1 will determine the structural requirements and functional consequences of serpin and proteinase binding to the receptor LRP. Specific Aim 2 will determine the structure of the mosquito serpin AFXa, its mechanism of inhibition of human factor Xa and the properties of the AFXa-Xa complex in relation to LRP binding and signaling. Specific Aim 3 will determine the conformational changes that occur in caspases, cathepsins and subtilisin-like proteinases upon formation of covalent complexes with serpins. We will use thermodynamic measurements of binding affinity and structural approaches of NMR spectroscopy, FRET and x-ray crystallography that we have successfully used in the previous grant period and, where appropriate, correlate our findings with functional consequences, through a collaboration with Dr. Dudley Strickland.