Structural and Functional Studies for Mitochondrial Protein Translocations

描述（由申请人提供）：线粒体膜上的蛋白质易位在线粒体生物发生中起着关键作用。蛋白质从细胞质溶胶到线粒体的转运是由外膜（TOM）复合体的转座酶和内膜（TIM）复合体的转座酶完成的。(1)在TOM复合体中，Tom70作为线粒体前体的受体，具有内部靶向信号。在我们的Tom70晶体结构中，Tom70的c端结构域形成一个大的口袋，可能代表线粒体前体的结合位点，Tom70的n端结构域可能起着门状口袋的作用。有趣的是，Tom70前体结合口袋的门控是由Hsp70/Hsp90结合调节的。Tom70-Hsp70/Hsp90复合物的晶体结构表明，Hsp70/Hsp90的c端EEVD基序可以使Tom70保持开放构象以接受线粒体前体。为了充分了解Tom70在Hsp70/Hsp90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鉴定了Tom70-Hsp70复合物的肽底物P70-8。Tom70- hsp70 EEVD基序-肽底物复合物的晶体结构将阐明Tom70在TOM转座子中作为分子伴侣结合的线粒体前体受体的机制。将进行基于结构的诱变研究来证实我们的假设。(2)在TIM23易位子中，Tim50作为受体引导具有n端前体到内膜蛋白通道TIM23进行易位。Tim50IMS可能与序列相互作用。Tim50IMS还可以与Tim23IMS相互作用，将前体传递到由Tim23的c端结构域形成的跨膜通道。Tim50IMS的晶体结构表明，一个突出的“发夹”可能代表Tim23的结合位点。在这个发夹附近，Tim50包含一个大凹槽，可能代表了前序列的结合位点。我们打算确定Tim50IMS-pre - sequence复合物、Tim50IMS-Tim23IMS复合物和Tim50IMS-Tim23IMS-pre - sequence复合物的晶体结构。(3) Tim23是Tim23转位子的主要成分，在线粒体内膜中形成必需的跨膜通道。为了揭示这种重要的膜蛋白转运线粒体前体的机制，我们建议确定Tim23的晶体结构。已知Tim23很难用许多系统来表达。在初步数据中，我们利用噬菌体展示文库筛选技术开发了酵母Tim23IMS的结晶伴侣。我们成功地在毕氏菌体系中利用“自裂”2A肽表达了Tim23与结晶伴侣的络合。平面脂质双分子层系统电生理分析表明重组Tim23具有良好的功能。

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.