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: 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
• 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' 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' -O-(3-thiotriphosphate); conformational alteration; endopeptidase Clp; interest; multicatalytic endopeptidase complex; 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 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的结构的实验室，这也是II型AAA+蛋白的第一个结构，其特征是具有两个连接的AAA+模块。我们还确定了CLPA的N末端结构域（N-域）的结构及其与CLP的复合物，CLP是一种衔接蛋白，在选择底物（N-End规则）中起作用以进行降解。我们分析了N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AAA+蛋白p97 ATPase突变体的许多N-D1片段的结构，这些结构在患有IBMPFD的患者中被鉴定出来。我们首次发现，当D1域与ATP结合时，突变蛋白的N末端结构域采取不同的构象。这与先前观察到的野生型酶中D1结构域的不变的N域构象与不变的ADP构象相反。观察到的从ADP-到ATPGAMMAS结合状态的过渡伴随着N-D1接头中的环向螺旋转换，并在p97的N末端区域中明显重新排序。我们的实验进一步表明，突变蛋白很可能对ADP的亲和力改变，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性N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。正在进行更多的生化和结构实验，以确认全长P97蛋白的发现。