Biochemistry of Energy-Dependent Protein Degradation

我们的研究主要集中在选择性蛋白质降解的机制和依赖于ATP的Lon和CLP酶的结构/功能关系上。LON和CLP在所有生物体中都存在，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。这些复杂的蛋白水解酶是具有至少两种活性的多域成分的集合。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展开酶活性。另一种成分是一种具有隔离活性部位的蛋白酶，可以通过狭窄的通道进入，该通道只允许延伸构象的蛋白质通过。ClpAP和ClpXP的电子显微镜为这些和其他依赖于ATP的蛋白酶提供了一个结构模型。ClpA是一个带有两个伴侣结构域的六角体。它与ClpP结合，ClpP是一个双层七聚体环，在环之间的内部腔中具有蛋白水解活性部位。ClpA也有一个内室，在那里蛋白质可以在转移到ClpP之前被展开或隔离。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 Di Xia博士合作，我们已经确定了ClpA的高分辨晶体结构和ClpA的N-末端结构域。ClpA的两个结构域都有折叠，使它们属于AAA超家族蛋白质，这是一组在所有活细胞中都具有重要的展开和分解活性的蛋白质。该结构提供了重要功能基序在ClpA中的位置和相互作用的详细信息，并加深了我们对其催化活性所必需的结构域组织、结构域相互作用和构象变化的理解。ClpA内的两个腔室具有很大程度上疏水的表面性质，但在沿六重轴的不同纬度处有正负电荷带。ClpA的N-末端结构域有一个新的折叠，使其能够与底物或适配器蛋白相互作用，从而介导其进入蛋白质底物。我们已经发现，其中一个这样的适配器，CLPS，其他人报道可以改变ClpA对底物的选择，只有当功能N-结构域存在时才作用于ClpA。生化研究揭示了CLP和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，这取决于底物或接头蛋白之间的竞争。对部分移位和降解的融合蛋白复合体的电子显微镜观察表明，底物从ATPase顶端表面的结合位置迁移到轴向通道上的位置，然后转移到复合体的内部。结合在复合体一侧的蛋白质可以被转移到ClpP中，而另一种底物仍然结合在另一端的伴侣上。ClpA的晶体结构揭示了两个ATP结构域之间的许多接触，这将使结构域之间能够进行通信，并提供一种从复合体两端相互转移底物的机制。人ClpP和人ClpX已得到表达和纯化。HClpP的晶体结构与E.coliClpP的晶体结构基本相同。HClpP具有在双层环的侧面上占据位置的C-末端延伸部分。这种延伸对hClpP的流体力学性质有很大的影响，并影响其基本的多肽酶活性。HClpX通过hClpP激活蛋白质降解，这是首次对哺乳动物ClpXP复合体显示出酶活性。人们正在利用在ClpP小室内隔离底物的能力来识别人和细菌CLP蛋白酶的活体靶标。

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.