Study of AAA proteins by X-ray protein crystallography

X射线蛋白质晶体学研究AAA蛋白质

• 批准号: 7592792
• 负责人: di s xia
• 金额: $ 11.87万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/7592792
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Adaptor Signaling Protein; Affinity; Alzheimer' s Disease; Binding; Biochemical; Biological Models; Cell Cycle Regulation; Chimeric Proteins; Complex; Coupling; Creutzfeldt-Jakob Syndrome; Crystallography; Enzymes; Escherichia coli; Family member; Frontotemporal Dementia; Goals; Human; Inclusion Bodies; Lead; Length; Link; Mechanics; Membrane Fusion; Methods; Molecular; Molecular Conformation; Motion; Movement; Myopathy; N Domain; N-terminal; Nucleotides; Numbers; Organism; Osteitis Deformans; Pathway interactions; Patients; Play; Polyubiquitin; Protein Family; Proteins; Resolution; Roentgen Rays; Role; Site; Structure; Structure-Activity Relationship; Time; Work; conformational alteration; endopeptidase Clp; interest; mutant; p97 ATPase; protein degradation; research study; unfoldase

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.

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+ 蛋白结构。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，这些突变体是在 IBMPFD 患者中发现的，并且被证明缺乏多聚泛素结合。我们首次发现，当 D1 结构域与 ATP 结合时，突变蛋白的 N 端结构域采取不同的构象，这与之前在野生型酶中 D1 结构域与 ADP 结合时观察到的构象形成鲜明对比。我们的实验进一步表明，突变蛋白很可能改变了对各种核苷酸的亲和力，从而导致观察到的不稳定构象改变。我们相信 p97 中新观察到的构象对于理解其功能至关重要。