Structural and Functional Studies for Mitochondrial Protein Translocations

描述(申请人提供)：蛋白质跨线粒体膜转位在线粒体生物发生中起关键作用。蛋白质从细胞质到线粒体的转运是通过外膜(TOM)复合体的转位酶和内膜(TIM)复合体的转位酶来实现的。(1)在TOM复合体中，Tom70是线粒体前体的受体，具有内靶向信号。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部位，而Tom70的N-末端结构域可能起到门控作用。有趣的是，Tom70前体结合口袋的门控受Hsp70/Hsp90结合的调节。Tom70-Hsp70/Hsp90复合体的晶体结构表明，Hsp70/Hsp90的C端EEVD基序可以保持Tom70的开放构象，以接收线粒体前体。为了全面了解Tom70与其多肽底物在Hsp70/Hsp90调控下的相互作用机制，我们通过噬菌体展示文库筛选获得了Tom70-Hsp70复合体的多肽底物P70-8。Tom70-Hsp70 EEVD基序-多肽底物复合体的晶体结构将阐明Tom70作为TOM易位子中分子伴侣结合的线粒体前体的受体的机制。我们将进行基于结构的突变研究来证实我们的假设。(2)在TIM23易位子中，TIM50作为受体引导N-末端的前体进入内膜蛋白通道TIM23进行转位。Tim50IMS可以与前序进行交互。Tim50IMS还可以与Tim23IMS相互作用，将前体递送到TIM23的C-末端结构域形成的跨膜通道。我们的TIM50IMS的晶体结构表明，一个突出的发夹可能代表TIM23的结合部位。在这个发夹附近，Tim50含有一个很大的凹槽，可能代表前序列的结合位点。我们打算确定Tim50IMS-前序列复合体、Tim50IMS-Tim23IMS复合体和Tim50IMS-Tim23IMS-前序列复合体的晶体结构。(3)TIM23是TIM23易位的主要成分，是线粒体内膜重要的跨膜通道。为了揭示这种重要的膜蛋白如何运输线粒体前体的机制，我们建议测定TIM23的晶体结构。众所周知，使用许多系统很难表达TIM23。在初步数据中，我们利用噬菌体展示文库筛选成功了酵母Tim23IMS的结晶伴侣。我们在毕赤酵母系统中成功地表达了TIM23与结晶性伴侣蛋白的复合体。平面脂质双层系统电生理分析表明，重组TIM23具有一定的功能。

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.