Study of AAA proteins by X-ray protein crystallography

我的实验室是第一个确定全长ClpA结构的实验室， II型AAA+蛋白的第一种结构，其特征在于具有两个串联的 连接AAA+模块。我们还确定了N-末端结构域（N-domain）的结构 ClpA及其与ClpS的复合物，ClpS是一种衔接蛋白，在选择中起作用。 底物（N-末端规则）降解。我们分析了N-结构域的结构， 确定了其与底物相互作用的潜在位点。最近，我的实验室 确定了人AAA+蛋白p97 ATP酶的许多N-D1片段的结构 突变体，其在患有IBMPFD的患者中鉴定。我们还观察到 突变蛋白的N-末端结构域采取不同构象的时间， D1结构域与ATP结合。这与先前观察到的不变N结构域相反 在野生型酶中，D1结构域中的ADP总是结合构象。我们 实验进一步表明，突变蛋白质很可能改变了对 导致观察到的不稳定构象改变的各种核苷酸。我们认为 在p97中观察到的新构象对于理解其功能至关重要。更 生物化学和结构实验正在进行，以证实这一发现， 全长p97蛋白。

My lab was the first to determine the structure of the full-length ClpA, which was also the first structure of type II AAA+ proteins, which are characterized by having two tandem connected AAA+ modules. We also determined the structures of the N-terminal domain (N-domain) of ClpA and its complex with ClpS, an adaptor protein that plays a role in selecting substrates (N-end rule) for degradation. We analyzed the structure of the N-domain and identified potential sites for its interaction with substrates. Recently, my lab has determined structures for a number of N-D1 fragments of the human AAA+ protein p97 ATPase mutants, which were identified in patients suffering from the IBMPFD. We found for the first time that the N-terminal domains of mutant proteins take a different conformation when the D1-domains are bound with ATP. This is in contrast to previously observed invariable N-domain conformation with invariably bound ADP in the D1 domains in the wild type enzyme. Our experiments further suggest that mutant proteins most likely have altered affinities for various nucleotides that lead to the observed erratic conformational alteration. We believe that the new observed conformation in p97 is critical for understanding its function. More experiments, both biochemical and structural, are being conducted to confirm this find with the full-length p97 protein.