Study of AAA proteins by X-ray protein crystallography

我的实验室是第一个确定全长ClpA结构的人，这也是II型AAA蛋白的第一个结构，其特征是有两个串联的AAA模块。我们还确定了ClpA的N-末端结构域(N-结构域)及其与CLPs的复合体的结构，CLPs是一种在选择底物(N-末端规则)方面起作用的适配蛋白。我们分析了N-结构域的结构，并确定了它与底物相互作用的潜在位置。最近，我的实验室已经确定了一些人类AAA蛋白p97 ATPase突变体的N-D1片段的结构，这些突变体是在IBMPFD患者中发现的。我们首次发现，突变蛋白的N-末端结构域与ATP结合时，其N-末端结构域呈现不同的构象。这与以前观察到的不变的N-结构域的构象相反，在野生型酶的D1域中总是结合ADP。我们的实验进一步表明，突变蛋白很可能改变了各种核苷酸的亲和力，从而导致观察到的不稳定构象改变。我们认为，p97中新观察到的构象对于理解其功能是至关重要的。更多的生化和结构实验正在进行中，以证实这一发现与全长的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.