The long-term goat of this proposal is to carry out X-ray crystallographic studies on Hsp40 to uncover the basic mechanisms by which Hsp40 interacts with non-native polypeptides and cooperates with Hsp70 to perform the molecular chaperone activity. We have proposed an """"""""anchoring and docking"""""""" model to illustrate the mechanisms for Hsp40 to interact with HspT0. To support this hypothesis, we intend to determine the crystal structure of the yeast type II Hsp40 Sis1 complexed with yeast Hsp70 Ssa1. We have constituted and crystallized the protein complex of Sis1 peptide-binding fragment and the Ssal C-terminal domain. Once solved, the complex crystal structure of Sis1 and Ssa1 will inform us how Hsp40 interacts with Hsp70 to carry out the non-native polypeptide delivery and assists Hsp in protein folding. The crystal structure of yeast type I Hsp40 Ydjl, complexed with the peptide substrate GWLYEIS, indicates that the pocket on the molecular surface of Ydjl may play important roles in peptide substrate binding. This pocket may exhibit significant plasticity to accommodate various sizes of hydrophobic side chains. We propose to determine the crystal structures of Ydjl peptide-binding fragment, complexed with peptide substrates GWWYEIS, and GWAYEIS. The crystal structures of Ydj1 and various peptide substrates may provide the structural basis of how Hsp40 adjusts itself to accommodate different peptide substrates. To reveal the mechanism by which Hsp40 primes the non-native polypeptide for the subsequent Hsp70 binding, we have designed a non-native polypeptide, Y1, by connecting two Hsp40 Ydj1 peptide substrates GWLYEIS with a flexible linker. The recombinant polypeptide Y1 showed a reasonable binding affinity to Hsp40 Ydjl. We intend to crystallize and determine the structure of the Ydjl complexed with the """"""""non-native polypeptide"""""""", Y1, to illustrate how Hsp40 may affect the conformation of the non-native polypeptide. Finally, we will conduct structure-based mutagenesis studies to test our proposed models for the mechanisms of Hsp40 chaperone actions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK056203-08
Application #
7113160
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Sechi, Salvatore
Project Start
1999-08-01
Project End
2009-07-31
Budget Start
2006-08-01
Budget End
2007-07-31
Support Year
8
Fiscal Year
2006
Total Cost
$311,504
Indirect Cost
Name
University of Alabama Birmingham
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Tao, Jiahui; Petrova, Kseniya; Ron, David et al. (2010) Crystal structure of P58(IPK) TPR fragment reveals the mechanism for its molecular chaperone activity in UPR. J Mol Biol 397:1307-15
Li, Jingzhi; Cui, Wenjun; Sha, Bingdong (2010) The structural plasticity of Tom71 for mitochondrial precursor translocations. Acta Crystallogr Sect F Struct Biol Cryst Commun 66:985-9
Hu, Junbin; Li, Jingzhi; Qian, Xinguo et al. (2009) The crystal structures of yeast Get3 suggest a mechanism for tail-anchored protein membrane insertion. PLoS One 4:e8061
Hu, Junbin; Li, Jingzhi; Qian, Xinguo et al. (2009) Preliminary X-ray crystallographic studies of yeast Get3. Acta Crystallogr Sect F Struct Biol Cryst Commun 65:489-91
Li, Jingzhi; Qian, Xinguo; Sha, Bingdong (2009) Heat shock protein 40: structural studies and their functional implications. Protein Pept Lett 16:606-12
Li, Jingzhi; Qian, Xinguo; Hu, Junbin et al. (2009) Molecular chaperone Hsp70/Hsp90 prepares the mitochondrial outer membrane translocon receptor Tom71 for preprotein loading. J Biol Chem 284:23852-9
Tao, Jiahui; Wu, Yunkun; Ron, David et al. (2008) Preliminary X-ray crystallographic studies of mouse UPR responsive protein P58(IPK) TPR fragment. Acta Crystallogr Sect F Struct Biol Cryst Commun 64:108-10
Li, Jingzhi; Wu, Yunkun; Qian, Xinguo et al. (2006) Crystal structure of yeast Sis1 peptide-binding fragment and Hsp70 Ssa1 C-terminal complex. Biochem J 398:353-60
Josyula, Ratnakar; Jin, Zhongmin; McCombs, Deborah et al. (2006) Preliminary crystallographic studies of yeast mitochondrial peripheral membrane protein Tim44p. Acta Crystallogr Sect F Struct Biol Cryst Commun 62:172-4
Wu, Yunkun; McCombs, Debbie; Nagy, Lisa et al. (2006) Preliminary X-ray crystallographic studies of yeast mitochondrial protein Tom70p. Acta Crystallogr Sect F Struct Biol Cryst Commun 62:265-7

Showing the most recent 10 out of 23 publications