酿酒酵母线粒体基质蛋白和的结构功能研究

酿酒酵母线粒体基质蛋白Mmf1和Mam33的结构功能研究

THE STRUCTURE AND FUNCTION STUDY OF S.CEREVISIAE MITOCHONDRIA MATRIX PROTEINS MMF1 AND MAM33

作者：蒲友光　　导师：周丛照

中国科学技术大学　　生物化学与分子生物学2011届博士

摘　要

YjgF/YER057c/UK114是一类在细菌、古生菌和真核细胞中高度保守的蛋白质家族，该家族蛋白质的分子量约为15 kDa，一级序列同源性高达47%～78%，但是它们的生物学功能却具有多样性。如人源蛋白hp14.5被认为是一个转录抑制因子，可以在大鼠网织红细胞裂解物系统内抑制蛋白质合成；大鼠中的rp14具有核糖核酸内切酶的活性；山羊中的UK114则有肿瘤抗原的活性。其他物种的同源蛋白的功能还包括钙蛋白酶活性（牛）、嘌呤调节活性（枯草芽孢杆菌）、光合作用的活性。

到目前为止，该家族已经有20多个蛋白的结构被解析，但它们的生理功能目前尚不清楚。这些已解析蛋白的总体结构都是三聚体，而且每两个单体之间形成大而深的沟槽，在这些沟槽中有6～9个非常保守的氨基酸，它们可以结合不同的配体，包括：苏氨酸、色氨酸、2-酮丁酸、乙二醇或丙酮。酿酒酵母中的同源蛋白是Mmf1，即线粒体基质因子（mitochondrial matrix factor），研究表明Mmf1可能参与异亮氨酸的生物合成或参与维持线粒体完整性等相关功能。

串联亲核纯化实验的结果显示Mmf1可以与线粒体酸性基质蛋白Mam33相互作用。因其是分子量约为33 kDa的酸性蛋白，又位于线粒体基质中，因此被命名为Mam33。它是在分离与细胞色素b2信号肽有相互作用的蛋白质中被发现的。Mam33基因的敲除会导致酵母在甘油培养基的条件下生长缓慢，但是对葡萄糖培养基中的生长则没有影响，这一结果显示Mam33可能参与了线粒体的氧化磷酸化作用。Mam33的同源蛋白在人源、硕大利什曼原虫、布氏锥虫和秀丽隐杆线虫中为三体或者四体，Mam33与它们的序列同源性约在24%～35%之间。人源的Mam33同源蛋白被称为是前体mRNA剪切因子结合蛋白，即p32。p32与维持线粒体氧化磷酸化相关，并通过与不同的配体蛋白结合来执行多种多样的功能。目前，一共有82个配体蛋白被鉴定出与酵母的Mam33有相互作用，其中23个是线粒体中的核糖体蛋白。因而，我们推测Mam33可能在Mmf1的帮助下参与线粒体中核糖体蛋白合成。

为了理解酿酒酵母中Mmf1与Mam33之间的相互作用关系，我们对这两个蛋白复合物进行了晶体学研究。虽然我们得到了Mmf1–Mam33复合物蛋白，但是未能长出晶体。但是我们成功解析了Mam33和Mmf1的单体结构，其分辨率分别为2.10 Å和1.74 Å。进而，我们进行了等温滴定实验、超速离心实验等生化实验。结果显示，Mmf1可以与Mam33组成摩尔比为2∶1的复合物。通过对Mmf1和Mam33的结构进行分析，我们发现Mmf1三体表面正电势与Mam33三体表面部分区域形成互补的表面电势，从而可能通过静电相互作用形成复合物。我们据此模拟出一个Mmf1–Mam33复合物的结构，它提供了两个蛋白之间可能的相互作用模式。

关键词　酿酒酵母　Mmf1　Mam33　线粒体氧化磷酸化　线粒体　晶体结构　蛋白质相互作用

Abstract

Members in the YjgF/YER057c/UK114 family are highly conserved in archaeo,bacteria and eukarya. All members of this have similar secondary structures and a molecular mass of approximately 15 kDa.　They share a highly conserved primary sequence with an identity from 47 to 78%,but possess diverse biological functions. The human homolog hp14.5 has been proposed as a putative translation inhibitor that can inhibit cell-free protein synthesis in the rabbit reticulocyte lysate system,whereas the rat homolog rp14.5 has endoribonuclease activity,and the goat homolog UK114 has tumor antigen activity. Biological functions of other homologs include the calpain activation in the bovine,the purine regulation activity in　Bacillus subtilis,and photosynthesis and chromoplastogenesis in plants. A line of evidences suggested that subtle sequence changes attribute the functional divergence among the members. To date,about twenty structures of this family have been determined,all of which adopt a trimeric structure with three clefts,each of which is characterized by 6–9 signature residues. These conserved clefts are able to bind diverse ligands,including L-threonine,L-serine,2-ketobutyrate,ethylene glycol,or propionate. However,the physiological significance of these members remains unclear. The homolog from the yeast Saccharomyces cerevisiae was named Mmf1,for mitochondrial matrix factor. Mmf1 was proposed to be involved in isoleucine biosynthesis and intact mitochondria maintenance.

Tandem affinity purification assays indicated that Mmf1 physically interacted with a mitochondrial acidic matrix protein Mam33,suggesting their putative functional relevance. Mam33 was found to be involved in sorting cytochrome b2 to the mitochondrial intermembrane space via binding to its signal peptide. Deletion of MAM33 gene would lead to a slower growth rate of yeast in glycerol medium but not in glucose medium,suggesting that Mam33 may participate in the mitochondrial oxidative phosphorylation. Mam33 was identified as a homo-trimer or tetramer and shared a sequence identity of approximately 24%～35% with the homologs from human,Leishmania major,Trypanosoma brucei,and　Caenorhabditis elegans. The human homolog of Mam33 was termed splicing factor 2-associated protein p32,which was proposed to be involved in the maintenance of mitochondrial oxidative phosphorylation. The human p32 executes diverse functions via binding to various partners. Up to date,a total of 82 protein partners were identified to have physical or genetic interactions with yeast Mam33. Among them,23 partners are mitochondrial ribosomal proteins. This led us to presume that,with the assistance of Mmf1,Mam33 may take part in mitochondrial ribosomal protein synthesis.

To figure out the structural insights into the interaction pattern between Mmf1 and Mam33,we attempted to solve the crystal structure of their complex. We purified the complex of Mam33–Mmf1,but we were unable to obtain its crystal. Nevertheless,we solved the crystal structures of individual Mmf1 and Mam33 at 1.74 and 2.10 Å,respectively.　In vitro biochemical assays demonstrated that Mmf1 and Mam33 can form a complex with a molar ratio of 2 to 1. Structural analysis revealed the positively charged surfaces of Mmf1 trimer are complementary to the negatively charged patches of Mam33 trimer. In addition,a docking model of Mmf1–Mam33 complex provides the structural basis for the putative binding mode of these two proteins.

Key words　Saccharomyces cerevisiae　Mmf1　Mam33　Mitochondrial oxidative phosphorylation Mitochondria　Crystal structure　Protein interaction