Biochemistry of Energy-Dependent Protein Degradation

我们的研究重点是选择性蛋白质降解的机制和atp依赖性的lonp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。这些复杂的蛋白酶是具有至少两种活性的多结构域组件的组装。一种组分结合蛋白质中的特定基序，并具有分子伴侣和蛋白质展开酶活性。另一组分是具有隔离活性位点的蛋白酶，该活性位点可以通过狭窄的通道进入，该通道只允许以扩展的构象通过蛋白质。电子显微镜下的ClpAP和ClpXP为这些和其他atp依赖性蛋白酶提供了结构模型。ClpA是具有两个伴侣结构域的六聚体。它与ClpP结合，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部腔室。ClpA也有一个内部腔室，蛋白质在转移到ClpP之前可以展开或隔离。我们在ClpA的结构测定方面取得了实质性的进展。与细胞生物学实验室的PI夏迪博士合作，我们确定了ClpA的高分辨率晶体结构和ClpA的n端结构域。ClpA的两个结构域都有折叠，使它们处于AAA蛋白超家族中，这是一组在所有活细胞中具有重要展开和拆卸活性的蛋白质。该结构提供了ClpA中重要功能基序的位置和相互作用的细节，并提高了我们对其催化活性重要的结构域组织，结构域相互作用和构象变化的理解。ClpA内部的两个腔室具有很大程度上疏水性的表面性质，但在沿六重轴的不同纬度上具有正负电荷带。ClpA的n端结构域具有一种新的褶皱，可能使其能够与底物或介导其进入蛋白质底物的衔接蛋白相互作用。我们发现一个这样的适配器ClpS，其他人报道它可以通过ClpA改变底物选择，只有当功能性n结构域存在时才作用于ClpA。生化研究揭示了ClpS与ClpA n结构域之间的直接相互作用。ClpA显然在不同的调控途径中发挥作用，这取决于底物或衔接蛋白之间的竞争。部分易位和降解的融合蛋白复合物的电子显微镜显示，底物从atp酶顶端表面的结合位点迁移到轴向通道上的一个位置，然后转移到复合物的内部。结合在复合体一侧的蛋白质可以转移到ClpP中，而另一个底物仍然结合在另一端的伴侣蛋白上。ClpA晶体结构揭示了两个ATP结构域之间的一些接触，这将使结构域之间的通信成为可能，并为复合物两端底物的相互易位提供了一种机制。表达并纯化了人ClpP和人ClpX。hClpP的晶体结构与大肠杆菌的晶体结构基本相同。hClpP具有c端延伸，其位于双层环的侧面。这种延伸对hClpP的水动力学特性有很大影响，并影响其基础肽酶活性。hClpX激活hClpP对蛋白质的降解，这是首次证实哺乳动物ClpXP复合物具有酶活性。在ClpP腔内隔离底物的能力被用来鉴定人和细菌Clp蛋白酶的体内靶点。

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.