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，这也是第一个具有两个AAA+模块的AAA+蛋白质结构。我们还确定了ClpA的N-末端结构域（N-结构域）及其与ClpS的复合物的结构，ClpS是一种衔接蛋白，在选择降解底物（N-末端规则）中起作用。我们分析了N-结构域的结构，并确定了其与底物相互作用的潜在位点。最近，我的实验室已经确定了许多人AAA+蛋白p97 ATP酶突变体的N-D1片段的结构，这些突变体在患有IBMPFD的患者中被鉴定出，并且被证明缺乏多聚泛素结合。我们首次发现，突变蛋白的N-末端结构域采取不同的构象时，D1-结构域与ATP结合，与以前观察到的构象时，D1结构域与ADP在野生型酶的形成鲜明对比。我们的实验进一步表明，突变蛋白质很可能改变了各种核苷酸的亲和力，导致观察到的不稳定的构象变化。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。

My lab was the first to determine the structure of the full-length ClpA, which was also the first structure of AAA+ proteins with two 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 and were shown to be deficient in polyubiquitin binding. 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, in sharp contrast with previously observed conformation when the D1 domains were bound with ADP in the wild type enzyme. Our experiments further suggest that mutant proteins most likely have altered the 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.