Structural and mechanistic studies of Hsp 100 protein

描述(由申请人提供)：本提案的目标是对Hspl 00进行结构研究，以揭示其作为分子伴侣发挥作用的机制。最近发现大肠杆菌Hsp1 00 ClpB通过解聚非天然多肽而发挥分子伴侣的作用。为了研究HplOO CIpB解聚非天然多肽的机制，我们建议确定ClpB的N末端结构域结构。研究表明，CIpB N末端结构域直接与非天然多肽相互作用，并在CIpB分子伴侣功能中发挥重要作用。我们已经结晶了ClpB的N-末端结构域，晶体的X射线衍射率为1.95A。在CIpB N-末端结构域的结构中，我们可以识别出一条多肽结合槽。因此，我们可以预测与Hsp1 00 CIpB结合的最小多肽长度。CIpB的高亲和力多肽底物将通过遗传和生物物理方法进行鉴定。我们将使CIpB N-末端结构域与其肽底物的络合物结晶。该复合体的晶体结构将为HplOO CIpB如何识别和结合非天然多肽提供基本的见解。为了揭示CIpB执行其ATPase活性的机制，我们建议用C-末端片段(D2C)来确定CIpB核苷酸结合域2的晶体结构。CIpB含有两个核苷酸结合区NbDi和Nbd2。我们解决了CIpB-NBDI的晶体结构，并结晶了与ATP或ADP络合的CIpB-D2C。为了测试HplOO CIpB伴侣功能的机制模型，我们将构建两组基于结构的CIpB突变体。一种是突变ClpB N-末端结构域中与结合肽密切相关的残基。这些突变体将通过肽结合分析和蛋白质折叠分析来测试功能的丧失。另一组突变体是为了支持所提出的CIpB ATPase活性的拉锯模型。我们将突变残基来阻止CIpB C-末端片段的构象变化。这些突变体将通过核苷酸结合分析、ATPase活性分析和蛋白质折叠分析进行功能测试。综上所述，这项建议涵盖了一项全面的研究，揭示了ClpB与非天然多肽相互作用并执行ATP水解以发挥分子伴侣功能的机制。

DESCRIPTION (provided by applicant): The goals of this proposal are to carry out structural studies on Hspl 00 to uncover the mechanisms by which it functions as a molecular chaperone. E. coli Hspl 00 ClpB was recently identified to act as a molecular chaperone by disaggregating non-native polypeptides. To investigate the mechanisms for HsplOO CIpB to disaggregate non-native polypeptides, we propose to determine ClpB N-terminal domain structure. The CIpB N-terminal domain has been shown to interact directly with non-native polypeptides and play essential roles in CIpB chaperone functions. We have crystallized the N-terminal domain of ClpB and the crystals diffracted X-ray to I .95A. In the structure of CIpB N-terminal domain, we may identify a peptide-binding groove. Therefore, we could predict the minimal length of peptides bound by Hspl 00 CIpB. The high affinity peptide substrates of CIpB will be identified by genetic and biophysical approaches. We will crystallize the complex of CIpB N-terminal domain with its peptide substrate. The crystal structure of the complex will provide fundamental insights on how HsplOO CIpB recognizes and binds the non-native polypeptides. To reveal the mechanisms for CIpB to carry out its ATPase activities, we propose to determine the crystal structure of CIpB nucleotide-binding domain 2 with the C-terminal fragment (D2C). CIpB contains two nucleotide-binding domains NBDI and NBD2. We have solved the crystal structure of CIpB NBDI and have crystallized CIpB D2C complexed with ATP or ADP. To test the models for the mechanisms for HsplOO CIpB chaperone functions, we will construct two sets of structure-based CIpB mutants. One is to mutate residues within ClpB N-terminal domain that are critically involved in binding peptides. These mutants will be tested for loss of functions by peptide binding assays and protein folding assays. The other set of mutants is to support the proposed "See-Saw" model for CIpB ATPase activity. We will mutate residues to disable the conformational changes of the CIpB C-terminal fragment. The mutants will be tested for functions by nucleotide binding assays, ATPase activity assays and protein folding assays. Collectively, this proposal covers a comprehensive study that reveals the mechanisms by which ClpB interacts with non-native polypeptides and perform ATP hydrolysis to function as a molecular chaperone.