Study of AAA proteins by X-ray protein crystallography

AAA+蛋白的结构和机制研究AAA+家族蛋白利用三磷酸腺苷水解产生的能量，在DNA复制过程中进行机械作用，通过重塑激活蛋白质，在蛋白质的复性和降解过程中发挥重要作用，从而在转录调节、膜融合、细胞周期控制等多种细胞活动中发挥重要作用。AAA+蛋白水解酶是一种结构和机械结构复杂的蛋白质，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，并有助于理解其他利用酶如ABC转运蛋白的功能。与LCB的M.Maurizi博士合作，我的实验室一直在研究原核生物ClpA蛋白展开酶的结构，这是CLP蛋白酶的一种成分，在许多生物体中是必不可少的，并且高度保守。我们确定了全长ClpA的结构，它是ClpAP复合体的调节成分。我们还确定了ClpA及其与CLPs的复合体的结构，CLPs是一种在选择降解底物方面起作用的适配蛋白。我们分析了ClpA的N-结构域的结构，并确定了它与底物相互作用的潜在位置。在与NCI的C.Li博士的合作下，我的实验室还结晶了几个人类AAA+蛋白p97突变体的N-D1片段，这些突变体缺乏泛素结合。我们测定了这些突变体的晶体结构，发现这些突变体蛋白的构象都与以前报道的不同，并且都与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的N-D1突变体的晶体，并测试这些核苷酸状态下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.