Biochemistry of Energy-Dependent (Intracellular) Protein

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存在于所有微生物和真核生物（包括哺乳动物）的细胞器中，它们有助于调节参与细胞控制的关键蛋白质的水平，并通过靶向错误折叠的蛋白质进行降解来帮助维持蛋白质质量控制。Lon和Clp是多结构域或多组分的组合，包括在双重对称蛋白酶复合物的两侧紧密结合的伴侣组分。伴侣蛋白（ClpA或ClpX或Lon的伴侣蛋白结构域）识别细胞内调控的选择性靶标中的特定基序，以及未折叠或错误折叠蛋白质的一般基序特征。伴侣蛋白展开蛋白质，从而溶解聚集体，分解复合物，或解开错误折叠的蛋白质，使它们可以被释放并允许重新折叠或被转移到相关的蛋白酶中进行降解。为了保护其他蛋白质免受不必要的损害，蛋白酶的活性位点（ClpP或Lon的蛋白酶结构域）被隔离在内部的水腔中，该水腔可通过狭窄的轴向通道通过蛋白酶进入，其亚基排列为六或七元环。在过去的一年里，我们实验室在四个主要领域取得了进展。首先，我们与NCI细胞生物学实验室的夏迪博士合作，继续确定ClpA的高分辨率晶体结构和ClpA的n端结构域。ClpA有两个atp酶结构域，它们都具有AAA超家族蛋白的折叠特征，AAA超家族蛋白是一组不同的能量依赖分子机器，在所有生物体中具有不同的展开和拆卸活性。该结构提供了对配体结合和催化残基相互作用的见解，并揭示了一种独特的结构域组织，其中D1和D2 atp酶结构域在ClpA六聚环周围交叉分布。功能受损突变的位置已经被映射到三维结构上，这被用作设计其他突变的模板，以探测ClpA的功能区域及其作用机制。n端结构域的结构揭示了一种新的褶皱，该褶皱可为底物和调节体内ClpA活性的衔接蛋白产生相互作用表面。n结构域也被发现有一个锌离子的结合位点，研究正在进行中，以确定金属离子在ClpA底物结合和展开中的作用。我们还获得了接头蛋白、ClpS和n结构域之间复合物的结构，这揭示了ClpS的n端17个氨基酸在阻断底物与ClpA结合方面的独特作用。其次，动力学生化研究和电子显微镜相结合表明，在转运步骤之前，有一个限速步骤（底物结合或展开），在这个步骤中，蛋白质从复合物的顶端表面转移到ClpP的降解室。我们的数据导致了一个模型，其中底物在对称复合体的一侧启动，而易位发生在另一侧，导致复合体两侧展开和易位的交替模式。第三，我们利用ClpA和ClpX识别的降解基序对应的肽来研究蛋白质识别位点的数量和性质。滴定量热法和荧光滴定法表明，单个肽与ClpA或ClpX的六聚体具有高亲和力。这种不寻常的化学计量表明，要么亚基中的结合位点重叠，一次只能占据一个，要么结合肽具有很强的负协同性，这样肽就会产生构象变化，降低相邻亚基上位点的结合亲和力。这些肽可以与伴侣交联，并且在ClpA的D1结构域内的一个位点已被指出是结合位点。具体的遗址鉴定工作正在进行中。关于底物结合和识别的全局信息已经从体内下拉试验中获得。通过过表达不降解蛋白质的ClpP突变体，可以通过质谱法捕获和鉴定底物。这些研究正在与唐纳德·亨特（弗吉尼亚大学）合作进行，并揭示了一些新的底物。鉴定不同底物或底物亚群之间的共同元素应提供有关基序范围的数据，揭示hClpX在促进ClpP双环结构稳定性方面的意想不到的作用。hClpP环在溶液中容易分离。除了ClpX结合时的构象变化外，ClpP的n端和催化活性位点的配体或残基也会影响四聚体的稳定性。这些数据提供了ClpP中环接触可能在催化过程中发生变化的第一个证据，并可能为底物在腔内的效应积累以及在过程降解后产物从腔中释放的可能机制提供了基础。我们正在接近完成hClpP的x射线晶体结构，它与E. coliClpP非常相似，但具有不同的n端构型。我们的结构表明，n端可能具有类似于蛋白酶体β亚基的n端延伸的门控功能。我们已经在大肠杆菌细胞中开发了一种人类ClpP的体内实验，并正在利用它来识别影响ClpX活性和与ClpX相互作用的突变，并正在扩展大肠杆菌模型系统，以包括人类ClpX，这将使ClpX功能改变突变的分离变得容易。

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 microorganisms and in the organelles of eukaryotes, including mammals, where they help regulate the levels of key proteins involved in cellular control and help maintain protein quality control by targeting misfolded proteins for degradation. Lon and Clp are multi-domain or multi-component assemblies comprising a chaperone component tightly associated on either side of a two-fold symmetric protease complex. The chaperone (ClpA or ClpX or the chaperone domain of Lon) recognizes specific motifs in selective targets for intracellular regulation and general motifs characteristic of unfolded or misfolded proteins. The chaperone unfolds proteins and thereby dissolves aggregates, disassemble complexes, or untangle misfolded proteins so they can either be released and allowed to refold or be translocated to the associated protease for degradation. To protect other proteins from unwanted damage, the active sites of the protease (ClpP or the protease domain of Lon) are sequestered in an internal aqueous chamber that is accessible by narrow axial channels through the protease, which has its subunits arranged six- or seven-membered rings. In the past year our laboratory has made progress in four major areas. First, we have (in collaboration with Dr. Di Xia, Laboratory of Cell Biology, NCI, continued to determine a high-resolution crystal structure for ClpA and the N-terminal domain of ClpA. ClpA has two ATPase domains, both of which have folds characteristic of the AAA super-family of proteins, a diverse group of energy-dependent molecular machines with various unfolding and disassembly activities in all organisms. The structure has provided insights into interactions of ligand-binding and catalytic residues and revealed a unique domain organization in which the D1 and D2 ATPase domains are interdigitated around the hexameric ring of ClpA. The positions of function impairing mutations have been mapped onto the 3D structure, which is being used as a template for the design of additional mutations to probe functional regions of ClpA and its mechanism of action. The structure of the N-terminal domain has revealed a novel fold that produces interaction surfaces for substrates and for an adaptor protein that modulates ClpA activity in vivo. The N-domain has also been found to have a binding site for a zinc ion and studies are underway to determine the function of the metal ion in ClpA substrate binding and unfolding. We have also obtained the structure of a complex between an adaptor protein, ClpS, and the N-domain, which reveals a unique role for the N-terminal 17 amino acids of ClpS in blocking substrate binding to ClpA. Second, a combination of kinetic biochemical studies and electron microscopy has shown that a rate-limiting step (substrate binding or unfolding) precedes the translocation step in which proteins are transferred from the apical surface of the complex to the degradation chamber of ClpP. Our data lead to a model in which substrates are primed on one side of the symmetrical complex while translocation occurs from the other side, resulting in an alternating mode of unfolding and translocation from the two sides of the complex. Third, we have used peptides corresponding to degradation motifs recognized by ClpA and ClpX to study the number and nature of protein recognition sites. Titration calorimetry and fluorescence titrations have shown that a single peptide binds with high affinity to a hexamer of ClpA or ClpX. This unusual stoichiometry suggests either that the binding sites in the subunits overlap so that only one can be occupied at a time or that there is strong negative cooperativity in binding peptides, such that a peptide produces a conformational change that decreases the binding affinity of sites on adjacent subunits. The peptides can be cross-linked to the chaperone and a site within the D1 domain of ClpA has been indicated as the site of binding. Specific identification of the site is underway. Global information about substrate binding and recognition has been obtained from in vivo pull-down assays. By overexpressing mutants of ClpP that do not degraded proteins, substrates can be trapped and identified by mass spectroscopy. These studies are going on in collaboration with Donald Hunt (U. Virginia) and have revealed a number of novel substrates. Identification of elements in common among the different substrates or subsets of substrates should provide data on the range of motifs revealed an unexpected effect of hClpX in promoting stability of the double ring structure of ClpP. hClpP rings readily separate in solution. In addition to conformational changes upon ClpX binding, ligands or residues at the N-terminus of ClpP and at the catalytic active site of ClpP have an influence on the stability of the tetradecamer. These data provide the first evidence that changes in ring contacts in ClpP may occur during catalysis and may provide a basis for effects substrate accumulation within the chamber and for a possible mechanism of product release from the chamber following processive degradation. We are nearing completion of an X-ray crystal structure of hClpP which is very similar to that of E. coliClpP but which has a different configuration of the N-terminus. Our structure suggests that the N-terminus may be in position to serve a gating function analogous to that described for the N-terminal extension of the proteasome beta subunits. We have developed an in vivo assay for human ClpP in E. coli cells and are exploiting it to identify mutations that affect activity and interaction with ClpX and are in the process of extending the E. coli model system to include human ClpX, which will allow the facile isolation of function altering mutations in ClpX.