Study of AAA proteins by X-ray protein crystallography

AAA+蛋白的结构和机理研究saaa +家族蛋白利用ATP水解产生的能量在DNA复制过程、蛋白质的重塑激活过程、蛋白质的再折叠和降解过程中进行机械工作，在转录调节、膜融合、细胞周期控制等多种细胞活动中发挥重要作用。AAA+蛋白酶是结构和机制复杂的蛋白质，其结构/功能关系反映了需要在亚分子水平上理解的重要生化原理，并有助于理解其他ATP利用酶（如ABC转运蛋白）的功能。我的实验室与LCB的M. Maurizi博士合作，一直致力于原核生物ClpA蛋白展开酶的结构研究，该酶是Clp蛋白酶的一个组成部分，在许多生物体中都是必不可少的，并且高度保守。我们确定了全长ClpA的结构，这是ClpAP复合物的调节成分。我们还确定了ClpA的n端结构域及其与ClpS的复合物的结构，ClpS是一种在选择降解底物中起作用的衔接蛋白。我们分析了ClpA的n域结构，并确定了其与底物相互作用的潜在位点。在与NCI的C. Li博士的合作下，我的实验室还结晶了一些缺乏泛素结合的人类AAA+蛋白p97突变体的N-D1片段。我们确定了这些突变体的晶体结构，发现突变蛋白的构象都与之前报道的不同，并且与ATP结合，这在D1结构域中没有观察到。我们正在进一步努力获得不同核苷酸结合状态下的n - d1突变体晶体，并在这些核苷酸状态下测试对泛素的生化结合亲和力。

Structural and mechanistic studies of AAA+ proteinsAAA+ family proteins utilize energy from ATP hydrolysis to perform mechanical works during the process of DNA replication, in protein activation by remodeling, and in protein refolding and degradation, through which they play important roles in various cellular activities, including transcription regulation, membrane fusion, cell cycle control, and so on. AAA+ proteases are structurally and mechanistically complex proteins, whose structure/function relationships reflect important biochemical principles that need to be understood at the sub-molecular level and would be beneficial to understanding functions of other ATP utilizing enzymes such as ABC transporters. In collaboration with Dr. M. Maurizi of LCB, my lab has been working on structural studies of the prokaryotic ClpA protein unfoldase, which is a component of the Clp proteases that are essential in many organisms and highly conserved. We determined the structure of full-length ClpA, the regulatory component of the ClpAP complex. We also determined the structures N-terminal domain of ClpA and its complex with ClpS, an adaptor protein that plays a role in selecting substrates for degradation. We analyzed the structures of the N-domain of ClpA and identified potential sites for its interaction with substrates. In collaboration with Dr. C. Li of NCI, my lab also crystallized the N-D1 fragments of a few human AAA+ protein p97 mutants, which are deficient in ubiquitin binding. We determined crystal structures of these mutants and found that the mutant proteins are all in a different conformation from previous reports and are bound with ATP, which was not observed for the D1 domain. We are furthering our effort to obtain crystals of N-D1mutants in different nucleotide binding states and testing biochemically binding affinities for ubiquitin under these nucleotide states.