Structural and mechanistic studies of Hsp 100 protein

描述（由申请人提供）：本提案的目标是对Hsp 100进行结构研究，以揭示其作为分子伴侣发挥功能的机制。E. coli Hsp 100 ClpB最近被鉴定为通过解聚非天然多肽而充当分子伴侣。为了研究HsplOO ClpB解聚非天然多肽的机制，我们提出确定ClpB N-末端结构域结构。ClpB N-末端结构域已被证明与非天然多肽直接相互作用，并在ClpB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，并且晶体的X射线衍射为1.95 A。在ClpB的N端结构域中，我们可以识别出一个肽结合沟。因此，我们可以预测由Hsp 100 ClpB结合的肽的最小长度。将通过遗传和生物物理方法鉴定ClpB的高亲和力肽底物。我们将使ClpB N-末端结构域与其肽底物的复合物结晶。复合物的晶体结构将提供关于HsplOO ClpB如何识别和结合非天然多肽的基本见解。为了揭示ClpB发挥ATP酶活性的机制，我们建议用C-末端片段（D2C）确定ClpB核苷酸结合结构域2的晶体结构。ClpB含有两个核苷酸结合结构域NBD 1和NBD 2。我们已经解决了ClpB NBDI的晶体结构，并已结晶ClpB D2C与ATP或ADP复合。为了测试HsplOO ClpB分子伴侣功能机制的模型，我们将构建两组基于结构的ClpB突变体。一种是突变ClpB N-末端结构域中关键参与结合肽的残基。将通过肽结合试验和蛋白质折叠试验检测这些突变体的功能丧失。另一组突变体是为了支持所提出的ClpB ATP酶活性的“See-Saw”模型。我们将突变残基以使ClpB C-末端片段的构象变化失效。将通过核苷酸结合试验、ATP酶活性试验和蛋白质折叠试验检测突变体的功能。总的来说，该提案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进行ATP水解以发挥分子伴侣作用的机制。

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.