Structural and Functional Studies for Mitochondrial Protein Translocations

线粒体蛋白质易位的结构和功能研究

• 批准号: 8396716
• 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
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8396716
• 关键词: Aging; Bacteriophages; Binding; Binding Sites; Biochemical Genetics; Biogenesis; C-terminal; Cancer Biology; Carrier Proteins; Cells; Cleaved cell; Collaborations; Complex; Crystallization; Cytosol; Data; Diabetes Mellitus; Dimensions; Germany; Goals; Human; In Vitro; Inner mitochondrial membrane; Integral Membrane Protein; Ion Channel; Libraries; Lipid Bilayers; Membrane; Membrane Proteins; Membrane Transport Proteins; Mitochondria; Mitochondrial Proteins; Modeling; Molecular Chaperones; Molecular Conformation; Mutagenesis; Mutation; N-terminal; Peptide Phage Display Library; Peptides; Phage Display; Pichia; Play; Protein translocation; Recombinants; Regulation; Research; Resolution; Roentgen Rays; Role; ST5 gene; Screening procedure; Signal Transduction; Structure; System; Universities; Work; Yeasts; base; in vivo; protein transport; receptor; translocase

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. PUBLIC HEALTH RELEVANCE: Protein translocations across mitochondria membranes play critical roles in mitochondria biogenesis. The long-term goal of this proposal is to carry out structural and functional studies on TOM and TIM complexes to uncover the basic mechanisms how the translocons facilitate the protein translocations across the mitochondria outer and inner membranes. The proposed research may have broad impacts on human aging, cancer biology and diabetes.

描述（由申请人提供）：蛋白质跨线粒体膜易位在线粒体生物发生中起关键作用。蛋白质从细胞质转运到线粒体是通过外膜转位酶（translocase of outer membrane，TOM）复合物和内膜转位酶（translocase of inner membrane，TIM）复合物完成的。(1)在TOM复合物中，Tom 70作为具有内部靶向信号的线粒体前体的受体发挥作用。在我们的Tom 70晶体结构中，Tom 70的C-末端结构域形成一个大口袋，其可能代表线粒体前体的结合位点，并且Tom 70的N-末端结构域可能起到门控口袋的作用。有趣的是，Tom 70的受体结合口袋的门控由Hsp 70/Hsp 90结合调节。Tom 70-Hsp 70/Hsp 90复合物的晶体结构表明，Hsp 70/Hsp 90的C-末端EEVD基序可以维持Tom 70处于开放构象以接收线粒体前体。为了深入了解Tom 70在Hsp 70/Hsp 90调控下与其肽底物相互作用的机制，我们通过噬菌体展示文库筛选，确定了Tom 70-Hsp 70复合物的肽底物P70-8。Tom 70-Hsp 70 EEVD基序-肽底物复合物的晶体结构将说明Tom 70如何在TOM易位子中作为分子伴侣结合的线粒体前体的受体发挥作用的机制。将进行基于结构的诱变研究以证实我们的假设。(2)在TIM 23转运子中，Tim 50作为受体引导具有N-末端前序列的前体到内膜蛋白通道Tim 23进行转运。Tim 50 IMS可以与前序列交互。Tim 50 IMS还可以与Tim 23 IMS相互作用，以将前体递送到由Tim 23的C-末端结构域形成的跨膜通道。我们的Tim 50 IMS的晶体结构表明，一个突出的<$-发夹可能代表Tim 23的结合位点。靠近这个发夹，Tim 50包含一个大的凹槽，可能代表前序列的结合位点。我们测定了Tim 50 IMS-前序列复合物、Tim 50 IMS-Tim 23 IMS复合物和Tim 50 IMS-Tim 23 IMS-前序列复合物的晶体结构。(3)TIM 23是TIM 23转位子的主要组成部分，它在线粒体内膜中形成必需的跨膜通道。为了揭示这一重要的膜蛋白如何转运线粒体前体的机制，我们建议确定Tim 23的晶体结构。已知Tim 23难以使用许多系统来表达。在初步的数据，我们已经开发了一个结晶伴侣酵母Tim 23 IMS使用噬菌体展示文库筛选。我们成功地在毕赤酵母系统中表达了与结晶伴侣复合的Tim 23。重组Tim 23是功能性的，如通过使用平面脂质双层系统的电生理分析所示。 公共卫生相关性：蛋白质跨线粒体膜易位在线粒体生物合成中起关键作用。该提案的长期目标是对TOM和TIM复合物进行结构和功能研究，以揭示translocons如何促进蛋白质跨线粒体外膜和内膜易位的基本机制。这项研究可能对人类衰老、癌症生物学和糖尿病产生广泛影响。