Study of AAA proteins by X-ray protein crystallography

X射线蛋白质晶体学研究AAA蛋白质

基本信息

批准号：
8175333
负责人：
di s xia
金额：
$ 25.2万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8175333
关键词：
ATP Hydrolysis ATP phosphohydrolase ATP-Binding Cassette Transporters Adaptor Signaling Protein Affinity Alzheimer&apos s Disease Binding Biochemical Biological Models Bone Diseases Complex Coupling Creutzfeldt-Jakob Syndrome Crystallography Degradation Pathway Enzymes Escherichia coli Family member Frontotemporal Dementia Goals Human Inclusion Bodies Lead Length Link Mechanics Methods Molecular Molecular Conformation Motion Movement Myopathy N Domain N-terminal Nucleotides Organism Pathway interactions Patients Play Protein Family Proteins Resolution Roentgen Rays Role Site Structure Structure-Activity Relationship Time Ubiquitin Work adenosine 5&apos -O-(3-thiotriphosphate)conformational alteration endopeptidase Clp interest multicatalytic endopeptidase complex mutant p97 ATPase protein degradation research study unfoldase

项目摘要

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.

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 II 型 AAA+ 蛋白的结构，其特征是具有两个串联连接的 AAA+ 模块。我们还确定了 ClpA 的 N 端结构域（N 域）及其与 ClpS 的复合物的结构，ClpS 是一种接头蛋白，在选择降解底物（N 端规则）方面发挥作用。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，我的实验室确定了人类 AAA+ 蛋白 p97 ATPase 突变体的许多 N-D1 片段的结构，这些片段是在 IBMPFD 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 末端结构域呈现不同的构象。这与之前观察到的野生型酶的 D1 结构域中恒定的 N 结构域构象和恒定结合的 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 连接子中的环到螺旋的转换以及 p97 N 末端区域的明显重新排序。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 亚基经历类似的核苷酸依赖性 N 结构域构象变化。我们相信 p97 中新观察到的构象对于理解其功能至关重要。正在进行更多的生化和结构实验，以用全长 p97 蛋白证实这一发现。