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）：跨线粒体膜的蛋白质易位在线粒体生物合成中发挥关键作用蛋白质从细胞质到线粒体的转运是由外膜易位酶（TOM）复合物和内膜易位酶进行的。 (TIM) 复合物。 (1) 在 TOM 复合物中，Tom70 充当具有内部靶向信号的线粒体前体的受体。在我们的 Tom70 晶体结构中，Tom70 的 C 端结构域形成一个大口袋可能代表线粒体前体的结合位点，Tom70 的 N 端结构域可能有意门控该口袋，Tom70 前体结合口袋的门控受 Hsp70/Hsp90/ 结合调节。 Hsp90复合物表明Hsp70/Hsp90的C端EEVD基序可以维持Tom70处于开放构象以完全接收线粒体前体。为了了解Tom70在Hsp70/Hsp90的调控下如何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鉴定了Tom70-Hsp70复合物的肽底物P70-8 Tom70-Hsp70 EEVD基序-肽底物的晶体结构。复合物将说明 Tom70 在基于结构的诱变研究中如何作为分子伴侣结合线粒体前体的受体发挥作用的机制。 (2)在TIM23易位子中，Tim50作为受体引导前体的N端前序列至内膜蛋白通道Tim23进行易位，Tim50IMS也可以与Tim23IMS相互作用以递送前体。 Tim23 的 C 端结构域形成的跨膜通道 我们的 Tim50IMS 晶体结构表明突出 ¿ -发夹可能代表 Tim23 的结合位点 ¿ -发夹，Tim50包含一个可能代表前序列结合位点的大凹槽，我们打算确定TIM23易位子中Tim50IMS-前序列复合物、Tim50IMS-Tim23IMS复合物和Tim50IMS-Tim23IMS-前序列复合物的晶体结构。线粒体内膜中的重要跨膜通道揭示其机制。 Tim23 是重要的膜蛋白转运线粒体前体，我们建议使用许多系统来确定 Tim23 的晶体结构。在初步数据中，我们使用噬菌体展示文库筛选开发了酵母 Tim23IMS 的结晶伴侣。我们已经在毕赤酵母系统中使用“自裂解”2A 肽成功表达了与结晶伴侣复合的 Tim23，重组 Tim23 具有功能性，如下所示。使用平面脂质双层系统进行电生理分析。 公共健康相关性：跨线粒体膜的蛋白质易位在线粒体生物合成中发挥着关键作用。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，以揭示易位子如何促进蛋白质跨膜易位的基本机制。拟议的研究可能对人类衰老、癌症生物学和糖尿病产生广泛的影响。