Structural and Functional Studies for Mitochondrial Protein Translocations

线粒体蛋白质易位的结构和功能研究

基本信息

批准号：
8844233
负责人：
BINGDONG SHA
金额：
$ 29.3万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-01 至 2017-04-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Protein translocations across mitochondria membranes play critical roles in mitochondria biogenesis. The protein transports from the cell cytosol to the mitochondria are carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complex. (1) In the TOM complex, Tom70 functions as the receptor for mitochondria precursors with internal targeting signals. In our Tom70 crystal structure, the C-terminal domain of Tom70 forms a large pocket which may represent the binding site for mitochondrial precursor and the N-terminal domain of Tom70 may function to gate the pocket. Interestingly, the gating of the precursor-binding pocket of Tom70 is regulated by Hsp70/Hsp90 binding. The crystal structure of Tom70-Hsp70/Hsp90 complex indicates that the C-terminal EEVD motifs of Hsp70/Hsp90 can maintain Tom70 in the open conformation for receiving mitochondrial precursor. To fully understand the mechanism how Tom70 interacts with its peptide substrate under the regulation of Hsp70/Hsp90, we have identified a peptide substrate P70-8 for Tom70-Hsp70 complex by phage display library screening. The crystal structure of Tom70-Hsp70 EEVD motif-peptide substrate complex will illustrate the mechanism how Tom70 functions as a receptor for the molecular chaperone-bound mitochondrial precursor in the TOM translocon. Structure-based mutagenesis studies will be performed to confirm our hypothesis. (2) In the TIM23 translocon, Tim50 functions as a receptor to guide the precursor with the N-terminal presequence to the inner membrane protein channel Tim23 for translocation. Tim50IMS may interact with the presequence. Tim50IMS can also interact with Tim23IMS to deliver the precursors to the transmembrane channel formed by the C-terminal domain of Tim23. Our crystal structure of Tim50IMS indicated a protruding ¿-hairpin may represent the binding site for Tim23. Close to this ¿-hairpin, Tim50 contains a large groove that may represent the binding site for the presequence. We intend to determine the crystal structures of Tim50IMS-presequence complex, Tim50IMS- Tim23IMS complex and Tim50IMS-Tim23IMS-presequence complex. (3) Tim23 represents the major component in TIM23 translocon and it forms the essential transmembrane channel in the mitochondrial inner membrane. To reveal the mechanism how this important membrane protein transports mitochondrial precursors, we propose to determine the crystal structure of Tim23. Tim23 has been known to be difficult to express using a number of systems. In preliminary data, we have developed a crystallization chaperone for yeast Tim23IMS using phage display library screening. We have successfully expressed Tim23 complexed with the crystallization chaperone using the "self-cleaving" 2A peptide in Pichia system. The recombinant Tim23 is functional as shown by electrophysiological analysis using planar lipid bilayer system.

描述（通过应用证明）：跨米孔氏菌在线粒体生物发生中起关键作用。 TOM70的端子域形成一个大口袋，它可能代表线粒体的结合位点，而TOM70的N末端域可能会通过HSP70/HSP90结合来调节口袋。表明HSP70/HSP90的C末端EEVD图案可以在开放同章中维持TOM70 OW TOM70与Tom70-HSP70-HSP70 eevd Motif-Peptide submitate tom70-HSP70晶体结构的HSP70/HSP90的法规，与ITHSTIDE替代替代替代相互作用Willux对TOM易位研究中的分子结合的内侧前体作为受体的机制将确认我们的假设（2）。 TIM50IM的内部蛋白质通道TIM23可能与TIM50IM相互作用。 -Hairpin可以代表TIM23的绑定位点。 - hairpin，tim50 presequence复合物，tim50ims-tim23ims复合物和tim50ims-tim23im-pressepequence（3）TIM23代表TIM23中的主要组成部分，并且它形成了基本的跨性别跨膜内在的内部粒子，内部蛋白质。我们提出使用许多系统的晶体结构，以确定Yeast Tim23ims使用噬菌体的phage e e e e e east tim23 Crystallization，使用Pichia系统中的“自切割” 2A肽的伴侣。