Biochemistry of Energy-Dependent (Intracellular) Protein

蛋白质生物化学部分的研究重点是细菌和人类细胞中蛋白质降解的功能和控制。细胞内蛋白质降解在控制重要的细胞调节蛋白水平方面起着关键作用，也是蛋白质质量控制系统的重要组成部分。胞浆中的大部分蛋白质降解是由依赖于ATP的蛋白酶执行的，这种酶是多组分的分子机器。这台机器的核心是一种由ATP驱动的蛋白质解折叠酶，它结合特定的蛋白质靶标，破坏其结构，并将未折叠的蛋白质转移到紧密相关的自我分隔的内肽酶的蛋白水解室中。我们的研究包括对来自大肠杆菌的依赖于ATP的CLP和Lon蛋白酶及其在人类线粒体中的同源物进行结构和生化分析。在过去的一年里，在几个领域取得了进展。基于我们的晶体结构，ClpP的活性部位有一个共价结合的多肽，我们已经产生了底物相互作用发生变化的突变体。底物具有移动结合模式，在这种模式下，多肽通过主链氢键相互作用，并可以在延伸的活性部位凹槽内滑动。只有P1残基具有明确的结合口袋。我们对S1口袋中的残基进行了突变，发现ClpP的活性受到了极大的影响。目前正在进行研究，以确定在S1口袋的表面属性发生变化的突变体中，切割的特异性是否受到影响。我们已经开始了对ClpP和ClpAP多肽酶和蛋白酶活性的快速动力学测量，以确定底物展开和易位的初始事件以及底物进入ClpP小室的机制。我们已经获得了ClpP环接触的动态变化的生化证据，这导致了十四聚体解离成两个七聚体环。这种解离应该反映在催化循环中发生的ClpP的构象变化，并可能与底物进入小室或降解后多肽产物从小室释放有关。CLPs是ClpA的接头，在体外可以抑制GFP-SsrA等底物的ClpA活性；然而，在体内，CLPs需要降解一类称为“N-端规则”的底物，这种底物含有非规范的氨基末端残基。模型N-末端规则底物在CLP存在的情况下被ClpA降解，并且这种降解需要作为CLP结合位点的N-结构域。我们正在通过使用ClpA和CLPS结合灭活的ClpP在体内捕获内源性N-端规则底物来识别它们。在体外，CLPs抑制ClpA活性，其化学计量比为每六聚体一个CLPs。我们正在使用冷冻电子显微镜(与A.C.Steven，NIAMS合作)和生化方法相结合的方法来确定CLPs对ClpA结构的影响以及与CLPs结合的N-结构域的分布。本实验室制备的特异性抗SsrA抗体已用于体内半衰期内源性SsrA标记蛋白的检测，并证明ClpXP在这些蛋白的降解中起主要作用。其他依赖于ATP的蛋白酶(Lon和ClpAP)可以降解SsrA标记的蛋白质，但只有在没有ClpX的情况下才能看到它们的作用。我们已经发现，许多蛋白质在大肠杆菌中的过度表达导致了带有SsrA标签的多种形式的蛋白质的大量产生。这些蛋白质大多被ClpXP降解，但当ClpXP活性受到影响时，这些蛋白质可以积累到显着水平。在我们旨在获得ClpA六聚体晶体结构的项目中，我们已经产生了在六聚体预期的亚基接触点引入半胱氨酸残基的ClpA突变体。

Research conducted in the Biochemistry of Proteins Section is focused on the function and control of protein degradation in bacterial and human cells. Intracellular protein degradation plays a critical part in controlling the levels of important cellular regulatory proteins and is an essential component of the protein quality control system as well. Most protein degradation within the cytosol is carried out by ATP-dependent proteases, which are multi-component, molecular machines. The heart of the machine is an ATP-driven protein unfoldase that binds a specific protein target, disrupts its structure, and translocates the unfolded protein into the proteolytic chamber of a tightly associated self-compartmentalized endopeptidase. Our studies encompass structural and biochemical analysis of the ATP-dependent Clp and Lon proteases from E. coli and their homologs in human mitochondria. In the last year, progress has been made in several areas. Based on our crystal structure of ClpP with a covalently bound peptide at the active site, we have generated mutants with altered substrate interactions. Substrates have a mobile binding mode in which polypeptides interact through main chain hydrogen bonding and can slide within an extended active site groove. Only the P1 residue has a defined binding pocket. We have mutated residues within the S1 pocket and found that the activity of ClpP is drastically affected. Studies are underway to determine whether the specificity of cleavage is affected in mutants in which the surface properties of the S1 pocket have been altered. We have initiated rapid kinetic measurements of ClpP and ClpAP peptidase and protease activity in order to determine the initial events in substrate unfolding and translocation and the mechanism by which substrates enter the ClpP chamber. We have obtained biochemical evidence of a dynamic change in ClpP ring contacts that results in dissociation of the tetradecamer into two heptameric rings. This dissociation should reflect a conformational change in ClpP that occurs during the catalytic cycle and could be related to substrate entry into the chamber or release of peptide products from the chamber after degradation. ClpS, an adaptor for ClpA, inhibits ClpA activity in vitro substrates such as GFP-SsrA; however, in vivo, ClpS is required for degradation of a class of substrates called "N-end rule" proteins, which have non-canonical amino terminal residues. Model N-end rule substrates are degraded by ClpA in the presence of ClpS and the N-domain, which is the binding site for ClpS, is required for this degradation. We are in the process of identifying endogenous N-end rule substrates by trapping them in vivo using ClpA and ClpS in combination with inactivated ClpP. In vitro, ClpS inhibits ClpA activity at a stoichiometry of one ClpS per hexamer. We are using a combination of cryo-electron microscopy (in collaboration with A. C. Steven, NIAMS) and biochemical methods to determine the effects of ClpS on the structure of ClpA and the distribution of the N-domains with ClpS bound. A specific anti-SsrA antibody prepared in our laboratory, has been used to measure the half-lives endogenous SsrA-tagged proteins in vivo and to demonstrate that ClpXP plays the major role in degradation of these proteins. Other ATP-dependent proteases (Lon and ClpAP) can degrade SsrA-tagged proteins but their contribution can be seen only in the absence of ClpX. We have found that over expression of many proteins in E. coli results in significant generation of multiple forms of the protein with SsrA-tags. These proteins are mostly degraded by ClpXP but can accumulate to significant levels when ClpXP activity is compromised. In our project aimed at obtaining the crystal structure of ClpA hexamers, we have generated ClpA mutants introducing cysteine residues at the subunit contact points expected in the hexamer.