CLP--AN ARCHETYPAL ATP DEPENDENT PROTEASE

异常蛋白质和许多短暂的调节因子是被一些依赖于ATP的蛋白酶机器降解的目标。这些机器在电子显微镜下都表现为大的低聚圆柱形复合体。这三个原型分别是26S蛋白酶体、CLP和LON。本提案侧重于代表三个原型中的两个的两个细菌蛋白酶机器(CLP和HsIUV)的结构和机制研究。CLP蛋白酶由一个蛋白分解核心ClpP和一个ATPase组分ClpA、ClpC或ClpX组成。HsIUV是由与265蛋白酶体的β亚基同源的蛋白水解组分HsIV(ClpQ)和类似CLP的ATPase组分Hsiu(ClpY)组成的杂交体。CLP ATPase亚基在体外和体内都具有伴侣活性，能够促进蛋白质的折叠/激活和降解。因此，这些ATPase可能是这些细胞通路中的一个重要决策点。除了205酶，HsIV是一类新的水解酶的定义成员之一，被称为NTN-水解酶(N-末端亲核体)，在NTN-水解酶中，末端苏氨酸残基充当亲核体。与Lon和26S蛋白酶体相比，CLP和HsIUV系统作为模型系统具有重大优势。在LON中，蛋白水解组分和ATPase组分都存在于单一的多肽链中，使得底物识别和蛋白降解是一个极其紧密耦合的过程。在我们的模型系统中，蛋白水解物和呈递的ATPase组分以稳定的同源低聚物的形式存在，它们在ATP存在的情况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，每个组分都是异源低聚物，因此必须使用标准的X射线技术来获得相位信息。在我们的系统中，组分是均低聚物和粗品，低分辨率，模型可以用来确定初始相，并通过利用非晶体对称性平均技术扩展到原子分辨率。我们已经开发了这项技术，并在已发表的ClpP结构中证明了其可行性。我们将再次使用这种方法来测定HsIV(ClpQ)，我们有3.0埃分辨率的数据。通过对称性平均，我们可以解决分子的结构和非晶体对称性的任何偏离可以在高分辨率(&gt；2.5埃)的电子密度图中看到。事实上，对ClpP结构的进一步改进确定了一种不对称性，这可能是ClpA优先结合到ClpP圆柱体的一个表面的原因。在此结构的基础上，我们提出了能量依赖的蛋白质降解的保守机制。这项建议的重点是确定蛋白分解机制(蛋白分解和ATPase)的单个组件的结构，并验证我们的假设。

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.