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.

我们的研究集中于选择性蛋白质降解的机制以及依赖ATP的LON和CLP蛋白酶的结构/功能关系。在所有生物体中都发现了LON和CLP，它们有助于调节重要的蛋白质水平并有助于蛋白质质量控​​制途径。这些复杂的蛋白酶是具有至少两种活性的多域成分的组件。一个成分结合蛋白质中的特定基序，并具有分子伴侣和蛋白展开酶活性。另一个成分是一个具有隔离活性位点的蛋白酶，该蛋白酶通过狭窄的通道可访问，该通道仅允许仅以扩展构象的形式传递蛋白质。 CLPAP和CLPXP的电子显微镜为这些和其他依赖ATP的蛋白酶提供了结构模型。 CLPA是具有两个伴侣域的己酰胺。它与Clpp（一种双层七聚环Clpp相关联），其蛋白水解活性位点位于环之间的内部腔室中。 CLPA还具有一个内部室，在转移到CLPP之前，可以在其中展开或隔离蛋白质。我们在CLPA的结构确定方面取得了重大进展。与细胞生物学实验室的PI Di Xia博士合作，我们确定了CLPA和CLPA的N末端结构域的高分辨率晶体结构。 CLPA的两个结构域都有折叠，将它们置于蛋白质的AAA超家族中，蛋白质是一组各种蛋白质，在所有活细胞中都具有重要的展开和拆卸活性。该结构提供了重要功能基序在CLPA中的位置和相互作用的细节，并改善了我们对域组织，域相互作用以及对其催化活性很重要的构象变化的理解。 CLPA内部的两个腔室的表面特性在很大程度上是疏水性的，但沿六倍轴的不同纬度的带有正电荷带。 CLPA的N末端结构域具有一种新颖的折叠，可以使其与底物或衔接蛋白相互作用，从而介导其访问其蛋白质底物的访问。我们发现，仅当存在功能性n个域时，其他人报告的一个这样的适配器CLP可以通过CLPA修改CLPA的底物选择。生化研究揭示了CLP与CLPA N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，具体取决于底物或衔接蛋白之间的竞争。与融合蛋白的复合物的电子显微镜部分被部分易位和降解，表明底物从ATPase的顶部表面上的结合位点迁移到轴向通道上的位置，然后转移到复合体的内部。结合在复合物的一侧的蛋白质可以迁移到CLPP中，而另一个底物则保持在另一端的伴侣蛋白上。 CLPA晶体结构揭示了两个ATP结构域之间的许多接触，这将使域之间的通信并为从复合物的任何一端提供了底物的相互易位的机制。人类CLPP和人类CLPX已被表达和纯化。 HCLPP的晶体结构实际上与大肠杆菌的晶体结构相同。 HCLPP具有C末端伸展，该延伸位置在双层环的侧面上占据位置。该延伸对HCLPP的流体动力特性具有很大的影响，并影响其基础肽酶活性。 HCLPX激活HCLPP的蛋白质降解，这是哺乳动物CLPXP复合物首次证明酶活性。隔离CLPP腔室内底物的能力正在被利用，以鉴定人类和细菌CLP蛋白酶的体内核心。