Study of AAA proteins by X-ray protein crystallography

我的实验室首先确定了全长ClpA的结构，这也是第一个具有两个串联连接AAA+模块的II型AAA+蛋白的结构。我们还确定了ClpA的n端结构域（N-domain）及其与ClpS的复合物的结构，ClpS是一种选择底物（n端规则）进行降解的衔接蛋白。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人类AAA+蛋白p97 atp酶突变体的N-D1片段的结构，这些突变体是在患有IBMPFD的患者中发现的。我们首次发现，当d1结构域与ATP结合时，突变蛋白的n端结构域具有不同的构象。这与之前在野生型酶中观察到的恒定n结构域构象与D1结构域的恒定结合ADP形成对比。观察到的从ADP-到atpgammas结合状态的转变伴随着N-D1连接体的环向螺旋转换以及p97 n端区域的明显重排序。我们的实验进一步表明，突变蛋白很可能对ADP具有改变的亲和力，从而导致观察到的不稳定构象改变。x射线散射实验表明野生型p97亚基经历了类似的核苷酸依赖的n结构域构象变化。我们认为p97中新观察到的构象对于理解其功能至关重要。更多的生物化学和结构实验正在进行，以证实全长p97蛋白的这一发现。

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 that 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. The observed transition from the ADP- to the ATPgammaS-bound state is accompanied by a loop-to-helix conversion in the N-D1 linker and by an apparent re-ordering in the N-terminal region of p97. Our experiments further suggest that mutant proteins most likely have an altered affinity for ADP that lead to the observed erratic conformational alteration. X-ray scattering experiments suggest that wild-type p97 subunits undergo a similar nucleotide-dependent N-domain conformational change. 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.