CLP--AN ARCHETYPAL ATP DEPENDENT PROTEASE

异常蛋白质和许多短寿命的调节剂的目标降解的ATP依赖的蛋白酶机器的数量。这些机器在电子显微镜下都表现为大的低聚圆柱形复合物。三种原型是26 S蛋白酶体、Clp和Lon。该建议的重点是两个细菌蛋白酶机器（CLP和HsIUV），代表三个原型中的两个的结构和机制的研究。Clp蛋白酶由蛋白水解核心ClpP和ATP酶组分ClpA、ClpC或ClpX组成。HsIUV是包含与265蛋白酶体的β型亚基同源的蛋白水解组分HsIV（ClpQ）和Clp样ATP酶组分HsIU（ClpY）的杂合体。Clp ATP酶亚基在体外和体内均表现出分子伴侣活性，并且能够促进蛋白质的折叠/活化和降解。因此，这些ATP酶可能是这些细胞通路中的重要决定点。除了205蛋白酶，HsIV是一类新的水解酶的定义成员之一，称为Ntn-水解酶（N-末端亲核试剂），其中末端苏氨酸残基充当亲核试剂。作为模型系统，Clp和HsIUV系统相对于Lon和26 S蛋白酶体具有主要优势。在Lon中，蛋白水解和ATP酶组分都存在于单个多肽链中，使得底物识别和蛋白水解是一个非常紧密耦合的过程。在我们的模型系统中，蛋白水解和呈递ATP酶组分作为稳定的同源寡聚体存在，其在ATP存在下瞬时缔合以降解蛋白质。这使我们能够分离，表达和操纵每个组件，并单独确定结构。在26 S蛋白酶体中，每个组分都是异源寡聚的，因此必须使用标准X射线技术获得定相信息。在我们的系统中，组件是同质寡聚和原油，低分辨率，模型可以用来确定初始阶段和扩展到原子分辨率，通过利用非晶体对称平均技术。我们已经开发了这种技术，并证明了它的可行性，在已公布的结构ClpP。我们将再次使用这种方法测定HsIV（ClpQ），我们有3.0埃分辨率的数据。通过对称平均，我们可以解决结构和任何偏离的分子从非晶体对称性可以看到在高分辨率（>2.5埃）的电子密度图。事实上，ClpP结构的进一步细化确定了一种不对称性，这可能是ClpA优先结合到ClpP圆柱体的一个面上的原因。在此基础上，我们提出了一个能量依赖性蛋白质水解的保守机制。该建议侧重于确定蛋白水解机制（蛋白水解和ATP酶）的各个组成部分的结构，并测试我们的假设。

Abnormal proteins and many short-lived regulators are targeted for degradation by a number of ATP-dependent protease machines. These machines all appear as large oligomeric cylindrical Complexes in electron micrographs. The three archetypes are the 26S proteasome, Clp and Lon. This proposal focuses on structural and mechanistic studies of two bacterial protease machines (Clp and HsIUV) that represent two of the three archetypes. The Clp protease is composed of a proteolytic core, ClpP and an ATPase component, ClpA, ClpC or ClpX. HsIUV is a hybrid comprising a proteolytic component, HsIV (ClpQ) that is homologous to the beta-type subunits of the 265 proteasome, and a Clp- like ATPase component, HsIU (ClpY). The Clp ATPase subunits exhibit chaperone activity both in vitro and in vivo and are capable of facilitating both the folding/activation and the degradation of proteins. As such, these ATPases may be an important decision point in these cellular pathways. In addition to the 205 protease, HsIV, is one of the defining members of a new class of hydrolytic enzymes, termed the Ntn-hydrolases (N-terminal nucleophile), in which the terminal threonine residue acts as the nucleophile. The Clp and HsIUV systems have major advantages over Lon and the 26S proteasome as a model system. In Lon, both the proteolytic and ATPase components reside with in a single polypeptide chain making substrate recognition and proteolysis an extremely tightly coupled process. In our model systems, the proteolytic and presenting ATPase components exist as stable homo-oligomers that associate transiently in the presence of ATP to degrade proteins. This allows us to separate, express and manipulate each component and determine the structures individually. In the 26S proteasome, each component is hetero-oligomeric and thus phasing information must be gained using standard x-ray techniques. In our systems, the components are homo-oligomeric and crude, low resolution, models can be used to determine initial phases and extended to atomic resolution by exploiting non-crystallographic symmetry averaging techniques. We have developed this technique and demonstrated its viability in the published structure of ClpP. We will use this approach again in the determination of HsIV(ClpQ) for which we have data to 3.0 Angstroms resolution. By symmetry averaging we can solve the structure and any deviations of the molecules from noncrystallographic symmetry can be seen in the electron density maps at high resolution (>2.5 Angstroms). Indeed, further refinement of the ClpP structure identified an asymmetry that may account for the preferential binding of ClpA to one face of the ClpP cylinder. On the basis of this structure, we have proposed a conserved mechanism for energy-dependent proteolysis. This proposal focuses on determining the structures of individual components of the proteolytic machinery (both proteolytic and ATPase) and testing our hypothesis.