CLP--AN ARCHETYPAL ATP DEPENDENT PROTEASE

异常蛋白和许多短寿命调节因子是一些atp依赖性蛋白酶机器降解的目标。这些机器在电子显微照片中都表现为大的低聚圆柱形复合物。这三种原型是26S蛋白酶体、Clp和Lon。本提案着重于两种细菌蛋白酶机器（Clp和HsIUV）的结构和机制研究，它们代表了三种原型中的两种。Clp蛋白酶由蛋白水解核心ClpP和atp酶组分ClpA、ClpC或ClpX组成。HsIV是一种杂交种，包括与265蛋白酶体的β型亚基同源的蛋白水解成分HsIV （ClpQ）和类似Clp的atp酶成分HsIU （ClpY）。Clp atp酶亚基在体外和体内均表现出伴侣活性，能够促进蛋白质的折叠/激活和降解。因此，这些atp酶可能是这些细胞途径中的重要决策点。除了205蛋白酶，HsIV是一类新的水解酶的定义成员之一，被称为n端亲核酶（n端亲核酶），其中末端苏氨酸残基充当亲核试剂。Clp和hhuv系统比Lon和26S蛋白酶体作为模型系统有很大的优势。在Lon中，蛋白水解和atp酶组分都位于单个多肽链中，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质低聚物存在，在ATP存在下短暂结合以降解蛋白质。这使我们能够分离、表达和操作每个组件，并单独确定结构。在26S蛋白酶体中，每个组分都是异聚寡聚的，因此必须使用标准x射线技术获得相位信息。在我们的系统中，组分是同质寡聚的、粗糙的、低分辨率的，模型可以用来确定初始相，并通过利用非晶体对称平均技术扩展到原子分辨率。我们已经开发了这种技术，并在已发表的ClpP结构中证明了它的可行性。我们将再次使用这种方法来确定HsIV(ClpQ)，我们有3.0埃分辨率的数据。通过对称平均，我们可以求解分子的结构，并且在高分辨率（> - 2.5埃）的电子密度图中可以看到分子与非晶体对称的任何偏差。事实上，对ClpP结构的进一步改进发现了一种不对称，这可能解释了ClpA优先结合到ClpP圆柱体的一面。基于这种结构，我们提出了一种能量依赖性蛋白水解的保守机制。该建议的重点是确定蛋白水解机制（包括蛋白水解和atp酶）的单个组分的结构，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.