STRUCTURAL AND FUNCTIONAL STUDIES OF HSP40

HSP40 的结构和功能研究

• 批准号: 8000171
• 负责人: BINGDONG SHA
• 金额: $ 8.5万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8000171
• 关键词: Affect; Affinity; Amino Acids; Antibodies; Binding; Biological Assay; C-terminal; Calorimetry; Cell physiology; Cells; Complex; Crystallization; Data; Dimerization; Docking; Exhibits; Goals; Goat; Grant; In Vitro; Length; Leucine; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Mutagenesis; Mutate; Mutation; Peptide Fragments; Peptide Phage Display Library; Peptides; Play; Progress Reports; Recombinants; Roentgen Rays; Role; Screening procedure; Side; Source; Stretching; Structure; Surface; Synchrotrons; Testing; Titrations; Work; Yeasts; base; design; flexibility; heat-shock proteins 40; in vivo; insight; loss of function; meetings; mutant; polypeptide; protein complex; protein folding; synthetic peptide

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.

描述(申请人提供)：这项建议的长期目标是对Hsp40进行X射线结晶学研究，以揭示Hsp40与非天然多肽相互作用并与Hsp70合作执行分子伴侣活性的基本机制。我们提出了一个“锚定和对接”模型来解释Hsp40与HspT0相互作用的机制。为了支持这一假设，我们打算确定酵母II型Hsp40Sis1与酵母Hsp70Ssa1的复合体的晶体结构。我们构建并结晶了Sis1肽结合片段和SSAL C-末端结构域的蛋白质复合体。一旦解决，Sis1和Ssa1的复杂晶体结构将告诉我们Hsp40如何与Hsp70相互作用进行非天然多肽的传递，并帮助HSP进行蛋白质折叠。I型酵母Hsp40Ydjl与多肽底物GWLYEIS络合的晶体结构表明，Ydjl分子表面的口袋可能在多肽底物结合中起重要作用。这个口袋可能表现出显著的可塑性，以适应不同大小的疏水侧链。我们建议确定Ydjl肽结合片段与多肽底物GWWYEIS和GWAYEIS络合的晶体结构。Ydj1和各种多肽底物的晶体结构可能为Hsp40如何调节自身以适应不同的多肽底物提供结构基础。为了揭示Hsp40为随后的Hsp70结合而启动非天然多肽的机制，我们设计了一个非天然多肽Y1，通过柔性连接子连接两个Hsp40 Ydj1肽底物GWLYEIS。重组多肽Y1与Hsp40Ydj1具有良好的结合亲和力。我们打算结晶和确定Ydjl与“非天然多肽”Y1的络合物的结构，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突变研究，以测试我们提出的Hsp40伴侣作用机制的模型。

项目成果

The crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 reveals novel dimerization motif for Hsp40.

• DOI: 10.1016/j.jmb.2004.12.040
• 发表时间: 2005-03
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Yunkun Wu;Jingzhi Li;Zhongmin Jin;Zhengqing Fu;B. Sha

The crystal structures of yeast Get3 suggest a mechanism for tail-anchored protein membrane insertion.

• DOI: 10.1371/journal.pone.0008061
• 发表时间: 2009-11-30
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Hu J;Li J;Qian X;Denic V;Sha B
• 通讯作者: Sha B

Preliminary X-ray crystallographic studies of yeast Get3.

酵母 Get3 的 X 射线晶体学初步研究。

• DOI: 10.1107/s1744309109012317
• 发表时间: 2009
• 期刊: Acta crystallographica. Section F, Structural biology and crystallization communications
• 作者: Hu,Junbin;Li,Jingzhi;Qian,Xinguo;Jin,Zhongmin;Fu,Zhengqing;Sha,Bingdong
• 通讯作者: Sha,Bingdong

Cloning, expression, purification and preliminary X-ray crystallographic studies of Escherichia coli Hsp100 ClpB nucleotide-binding domain 1 (NBD1).

大肠杆菌 Hsp100 ClpB 核苷酸结合域 1 (NBD1) 的克隆、表达、纯化和初步 X 射线晶体学研究。

• DOI: 10.1107/s0907444901007296
• 发表时间: 2001
• 期刊: Acta crystallographica. Section D, Biological crystallography
• 作者: Li,J;Sha,B
• 通讯作者: Sha,B

Peptide substrate identification for yeast Hsp40 Ydj1 by screening the phage display library.

• DOI: 10.1251/bpo90
• 发表时间: 2004
• 期刊: Biological procedures online
• 影响因子: 6.4
• 作者: Li J;Sha B
• 通讯作者: Sha B