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）：线粒体膜的蛋白质易位在线粒体生物发生中起关键作用。蛋白质从细胞胞质到线粒体的蛋白质通过外膜（TOM）复合物的易位酶和内膜（TIM）复合物的易位酶进行。 （1）在Tom复合物中，TOM70充当带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末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末端结构域可能会在口袋中发挥作用。有趣的是，TOM70的前体结合口袋的门控受HSP70/HSP90结合调节。 TOM70-HSP70/HSP90复合物的晶体结构表明HSP70/HSP90的C末端EEVD基序可以将TOM70维持在开放构象中，以接收线粒体前体。为了充分理解在HSP70/HSP90调节下TOM70与其肽底物相互作用的机制，我们通过噬菌体显示库筛选确定了TOM70-HSP70复合物的肽底物P70-8。 TOM70-HSP70 EEVD基型肽底物复合物的晶体结构将说明TOM70在TOM ClresSocon中如何充当分子伴侣结合的线粒体前体的受体的机制。将进行基于结构的诱变研究以确认我们的假设。 （2）在TIM23转运中，TIM50充当受体，可引导前体具有N末端的前体，直达内膜蛋白通道TIM23进行易位。 TIM50IM可能会与presequence相互作用。 Tim50IMS can also interact with Tim23IMS to deliver the precursors to the transmembrane channel formed by the C-terminal domain of Tim23.我们的TIM50IM的晶体结构表明，突出的`-Hairpin可能代表TIM23的结合位点。靠近此�-HAIRPIN，TIM50包含一个大的凹槽，可能代表presequence的结合位点。我们打算确定TIM50IMS-PRESEQUENCE复合物，TIM50IMS-TIM23IMS复合物和TIM50IMS-TIM23IMS-PRESEQUENCE-PRESEQUENCE复合物的晶体结构。 （3）TIM23代表TIM23转运的主要成分，它在线粒体内膜中形成了必需的跨膜通道。为了揭示该重要的膜蛋白如何转运线粒体前体的机制，我们建议确定TIM23的晶体结构。众所周知，TIM23很难使用许多系统表达。在初步数据中，我们使用噬菌体显示库筛选为酵母TIM23IM开发了结晶伴侣。我们已经成功地表达了TIM23与Pichia系统中的“自裂” 2A肽与结晶伴侣复合的tim23。重组TIM23具有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：线粒体膜之间的蛋白质易位在线粒体生物发生中起关键作用。该提案的长期目标是对TOM和TIM复合物进行结构性和功能研究，以揭示易位机制如何促进跨线粒体外部和内膜的蛋白质易位。拟议的研究可能会对人类衰老，癌症生物学和糖尿病产生广泛的影响。