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的作用机制。最近鉴定出大肠杆菌 Hspl 00 ClpB 通过解聚非天然多肽充当分子伴侣。为了研究Hsp100 CIpB解聚非天然多肽的机制，我们建议确定ClpB N端结构域结构。 CIpB N 端结构域已被证明可直接与非天然多肽相互作用，并在 CIpB 伴侣功能中发挥重要作用。我们已经结晶了 ClpB 的 N 端结构域，并且该晶体的 X 射线衍射为 I .95A。在CIpB N-末端结构域的结构中，我们可以识别肽结合沟。因此，我们可以预测 Hspl 00 CIpB 结合的肽的最小长度。 CIpB 的高亲和力肽底物将通过遗传和生物物理方法进行鉴定。我们将结晶 CIpB N 末端结构域与其肽底物的复合物。该复合物的晶体结构将提供关于Hsp100 CIpB如何识别和结合非天然多肽的基本见解。为了揭示 CIpB 执行 ATPase 活性的机制，我们建议确定 CIpB 核苷酸结合域 2 与 C 端片段 (D2C) 的晶体结构。 CIpB包含两个核苷酸结合结构域NBDI和NBD2。我们解析了CIpB NBDI的晶体结构，并结晶了与ATP或ADP复合的CIpB D2C。为了测试 Hspl00 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 端结构域内与结合肽至关重要的残基。将通过肽结合测定和蛋白质折叠测定来测试这些突变体的功能丧失。另一组突变体是支持所提出的 CIpB ATPase 活性的“See-Saw”模型。我们将突变残基以禁用 CIpB C 末端片段的构象变化。将通过核苷酸结合测定、ATP酶活性测定和蛋白质折叠测定来测试突变体的功能。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 水解以充当分子伴侣的机制。

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.