Structural and mechanistic studies of Hsp 100 protein

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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.