Structural examination of serpin-protein interactions

描述(由申请人提供)：尽管kunitz、Kazal和Bowman-Birk家族的蛋白质丝氨酸蛋白酶抑制剂可以通过锁和钥匙类型的相互作用来抑制目标蛋白酶，但现在x射线结晶学、核磁共振光谱和FRET表明，Serpins使用了一种显著的基于构象变化的机制来抑制一些相同的丝氨酸蛋白酶。这种机制涉及到丝氨酸和蛋白酶的变化，将丝氨酸反应中心环(RCL)插入到其自身的b-折叠A的中心，并将超过70A的蛋白酶从其初始对接位置移位到丝氨酸的远端，伴随而来的是蛋白酶活性部位的扭曲，以及当蛋白酶到达其最终位置时，很大一部分蛋白酶被压碎。虽然这些研究为蛇类如何抑制丝氨酸蛋白酶提供了结构性的解释，但它们并没有解释为什么要使用这种复杂的、容易出错的机制。一种未经证实的假设是，丝氨酸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LRP等受体识别和信号传递，还是通过丝氨酸-蛋白酶复合体中的切割而使蛋白酶无法恢复。我们提出了三个特定的目标，这三个目标结合在一起将能够评估完整的蛋白酶转位、蛋白酶粉碎序列在表达使蛇类在许多情况下成为首选的一类蛋白酶抑制剂的功能方面的重要性。具体目标1将确定丝氨酸和蛋白酶与受体LRP结合的结构要求和功能后果。具体目标2将确定蚊子丝氨酸AFXa的结构，其抑制人类Xa因子的机制，以及AFXa-Xa复合体与LRP结合和信号转导的性质。具体目标3将确定caspase、组织蛋白和类枯草杆菌蛋白水解酶在与蛇毒形成共价复合体时发生的构象变化。我们将使用结合亲和力的热力学测量以及我们在上一次赠款期间成功使用的核磁共振光谱、FRET和X射线结晶学的结构方法，并在适当的情况下，通过与Dudley Strickland博士的合作，将我们的发现与功能结果相关联。

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.