STRUCTURAL AND FUNCTIONAL STUDIES OF HSP40

描述（由申请人提供）：本提案的长期目标是对Hsp40进行X射线晶体学研究，以揭示Hsp40与非天然多肽相互作用并与Hsp70协同发挥分子伴侣活性的基本机制。我们提出了一个“锚定和对接”的模型来说明的机制，热休克蛋白40与热休克蛋白T0。为了支持这一假设，我们打算确定与酵母Hsp70 Ssa1复合的酵母II型Hsp40 Sis1的晶体结构。我们构建并结晶了Sis 1肽结合片段和Ssal C末端结构域的蛋白复合物。一旦解决，Sis1和Ssa1的复杂晶体结构将告诉我们Hsp40如何与Hsp70相互作用以进行非天然多肽递送并协助Hsp蛋白质折叠。与肽底物GWLYEIS复合的酵母I型Hsp40 Ydjl的晶体结构表明，Ydjl分子表面上的口袋可能在肽底物结合中起重要作用。该口袋可以表现出显著的可塑性以容纳各种尺寸的疏水侧链。我们建议确定Ydjl肽结合片段与肽底物GWWYEIS和GWAYEIS复合的晶体结构。Ydj1和各种肽底物的晶体结构可以提供Hsp40如何调节自身以适应不同肽底物的结构基础。为了揭示Hsp40引发非天然多肽用于随后的Hsp70结合的机制，我们通过用柔性接头连接两个Hsp40 Ydj1肽底物GWLYEIS来设计非天然多肽Y1。重组多肽Y1与Hsp40 Ydj1具有较好的结合活性。我们打算结晶和确定与“非天然多肽”Y1复合的Ydjl的结构，以说明Hsp40如何影响非天然多肽的构象。最后，我们将进行基于结构的诱变研究，以测试我们提出的模型的机制，热休克蛋白40分子伴侣行动。

DESCRIPTION (provided by applicant): 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.