Biochemistry of Energy-Dependent Protein Degradation

能量依赖性蛋白质降解的生物化学

• 批准号: 6558935
• 负责人: MICHAEL MAURIZI
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6558935
• 关键词: Escherichia coli; HeLa cells; active sites; adenosine triphosphate; adenosinetriphosphatase; bacterial proteins; bioenergetics; chemical stability; electron microscopy; endopeptidases; enzyme activity; enzyme complex; enzyme structure; enzyme substrate; enzyme substrate complex; gel filtration chromatography; human genetic material tag; intermolecular interaction; molecular chaperones; molecular cloning; protein degradation; protein structure function

Our research is focused on the mechanisms of selective protein degradation and the structure/function relationships of the ATP-dependent Lon and Clp proteases. Lon and Clp are found in all organisms, where they help regulate the levels of important proteins and contribute to protein quality control pathways. These complex proteases are assemblies of multi-domain components with at least two types of activity. One component binds specific motifs in proteins and has molecular chaperone and protein unfoldase activity. The other component is a protease with a sequestered active site that is accessible through narrow channels that permit passage of proteins only in an extended conformation. Electron microscopy of ClpAP and ClpXP has provided a structural model for these and other ATP-dependent proteases. ClpA is a hexamer with two chaperone domains. It associates with ClpP, a double-layered heptameric ring with proteolytic active sites located in an internal chamber between the rings. ClpA also has an internal chamber where proteins may be unfolded or sequestered prior to transfer to ClpP. We have made substantial progress in structure determination of ClpA. Working with Dr. Di Xia, a PI in the Laboratory of Cell Biology, we have determined a high resolution crystal structure for ClpA and the N-terminal domain of ClpA. Both domains of ClpA have folds that place them in the AAA super-family of proteins, a diverse group of proteins with important unfolding and disassembly activity in all living cells. The structure has provided details of the positions and interactions of important functional motifs in ClpA and has improved our understanding of the domain organization, domains interactions, and conformational changes that are important for its catalytic activity. The two chambers of within ClpA have surface properties that are largely hydrophobic, but with bands of positive and negative charges at different latitudes along the six-fold axis. The N-terminal domain of ClpA has a novel fold that may enable it to interact with substrates or adaptor proteins that mediate its access to protein substrates. We have found that one such adaptor, ClpS, which others reported could modify substrate selection by ClpA, acts on ClpA only when a functional N-domain is present. Biochemical studies reveal a direct interaction between ClpS and the ClpA N-domain. ClpA apparently functions in different regulatory pathways depending on competition between substrates or adaptor proteins. Electron microscopy of complexes with fusion protein that are partially translocated and degraded have shown that substrates migrate from a binding site on the apical surface of the ATPase to a position over an axial channel, and thereafter are transferred to the interior of the complex. Protein bound on one side of the complex can be translocated into ClpP while another substrate remains bound on the chaperone at the other end. The ClpA crystal structure reveals a number of contacts between the two ATP domains which would enable communication between the domains and provide a mechanism for reciprocal translocation of substrates from either end of the complex. Human ClpP and human ClpX have been expressed and purified. The crystal structure of hClpP is virtually identical to that of E. coliClpP. hClpP has a C-terminal extension which occupies a position on the lateral surface of the double-layered ring. This extension has a large effect on the hydrodynamic properties of hClpP and affects its basal peptidase activity. hClpX activates protein degradation by hClpP, the first time that enzymatic activity has been demonstrated for the mammalian ClpXP complex. The ability to sequester substrates within the ClpP chamber is being exploited to identify in vivotargets of both the human and bacterial Clp proteases.

我们的研究重点是选择性蛋白质降解的机制和ATP依赖的Lon和Clp蛋白酶的结构/功能关系。Lon和Clp存在于所有生物体中，它们有助于调节重要蛋白质的水平，并有助于蛋白质质量控制途径。这些复合蛋白酶是具有至少两种类型活性的多结构域组分的组装体。一种成分结合蛋白质中的特定基序，并具有分子伴侣和蛋白质解折叠酶活性。另一种组分是具有隔离活性位点的蛋白酶，该活性位点可通过仅允许蛋白质以延伸构象通过的狭窄通道进入。ClpAP和ClpXP的电子显微镜为这些和其他ATP依赖性蛋白酶提供了结构模型。ClpA是具有两个分子伴侣结构域的六聚体。它与ClpP相关联，ClpP是一种双层七聚体环，其蛋白水解活性位点位于环之间的内室中。ClpA还具有内室，其中蛋白质可以在转移到ClpP之前解折叠或隔离。我们在ClpA的结构测定方面取得了实质性进展。我们与细胞生物学实验室的PI Di Xia博士合作，确定了ClpA的高分辨率晶体结构和ClpA的N-末端结构域。ClpA的两个结构域都具有折叠，将它们置于AAA超家族蛋白质中，AAA超家族是在所有活细胞中具有重要的解折叠和分解活性的一组不同的蛋白质。该结构提供了详细的位置和重要的功能基序在ClpA的相互作用，并提高了我们的理解域组织，域的相互作用，和构象变化，是重要的催化活性。ClpA内的两个腔室具有很大程度上疏水的表面特性，但在沿着六重轴的不同纬度处具有正电荷和负电荷带。ClpA的N-末端结构域具有新的折叠，这可能使其能够与介导其进入蛋白质底物的底物或衔接蛋白相互作用。我们已经发现，一个这样的适配器，ClpS，其他人报道可以修改底物选择ClpA，作用于ClpA只有当一个功能性的N-结构域存在。生物化学研究揭示了ClpS和ClpA N-结构域之间的直接相互作用。ClpA显然在不同的调节途径中起作用，这取决于底物或接头蛋白之间的竞争。电子显微镜的复合物与融合蛋白的部分易位和降解表明，基板迁移从ATP酶的顶端表面上的结合位点的轴向通道上的位置，然后被转移到内部的复杂。结合在复合物一侧的蛋白质可以移位到ClpP中，而另一种底物在另一端保持结合在伴侣上。ClpA晶体结构揭示了两个ATP结构域之间的许多接触，这将使结构域之间的通信，并提供了一种机制，从复合物的任一端的底物的相互易位。人ClpP和人ClpX已经表达和纯化。hClpP的晶体结构与E. coliClpP. hClpP具有C-末端延伸，其占据双层环的侧表面上的位置。这种延伸对hClpP的流体动力学性质有很大的影响，并影响其基础肽酶活性。hClpX通过hClpP激活蛋白质降解，首次证明了哺乳动物ClpXP复合物的酶活性。将底物隔离在ClpP室内的能力被用于鉴定人和细菌Clp蛋白酶的体内靶标。