CLP--AN ARCHETYPAL ATP DEPENDENT PROTEASE

CLP--原型 ATP 依赖性蛋白酶

• 批准号: 2756779
• 负责人: John M Flanagan
• 金额: $ 34.69万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2756779
• 关键词: X ray crystallography; adenosine triphosphate; binding sites; bioenergetics; electron density; endopeptidases; enzyme activity; enzyme complex; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; hybrid enzyme; molecular chaperones; myosins; proteasome; proteolysis

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.

异常的蛋白质和许多短寿命调节剂都是由许多依赖ATP的蛋白酶机降解的。这些机器都显示为电子显微照片中的大型寡聚圆柱体复合物。三种原型是26S蛋白酶体，CLP和LON。该建议的重点是两种细菌蛋白酶机（CLP和HSIUV）的结构和机械研究，这些蛋白酶机器代表了三种原型中的两种。 CLP蛋白酶由蛋白水解核，CLPP和ATPase组件，CLPA，CLPC或CLPX组成。 HSIUV是一种杂种，包括与265个蛋白酶体的β型亚基同源的蛋白水解成分（CLPQ），以及类似于Clp的ATPase成分HSIU（Clpy）。 CLP ATPase亚基在体外和体内都表现出伴侣活性，并且能够促进蛋白质的折叠/激活和降解。因此，这些ATPases可能是这些细胞途径中的重要决策点。除205蛋白酶外，HSIV是一种新型水解酶的定义成员之一，称为NTN-氢酚酶（N末端核定剂），其中末端苏氨酸残基充当核粒子。与LON和26S蛋白酶体作为模型系统相比，CLP和HSIUV系统具有很大的优势。在LON中，蛋白水解和ATPase成分都位于单个多肽链中，从而使底物识别和蛋白水解成为非常紧密的过程。在我们的模型系统中，蛋白水解和呈现ATPase成分是作为稳定的同性恋物体存在的，它们在ATP存在下暂时关联以降解蛋白质。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26S蛋白酶体中，每个组件都是异弱的，因此必须使用标准的X射线技术获得相分信息。在我们的系统中，组件是同性化的和原油，低分辨率，模型可用于确定初始阶段，并通过利用非结晶对称性平均技术扩展到原子分辨率。我们已经开发了这种技术，并证明了其在CLPP发表的结构中的生存能力。我们将在确定HSIV（CLPQ）的确定中再次使用此方法，该数据将数据具有3.0 Angstroms分辨率。通过对称平均，我们可以在高分辨率（> 2.5盎司）的电子密度图中看到分子与非晶体密度对称性的任何偏差。实际上，对CLPP结构的进一步完善确定了一种不对称性，该不对称性可能解释了CLPA与CLPP圆柱体的一张面的优先结合。在这种结构的基础上，我们提出了一种保守的能量依赖性蛋白水解的机制。该提案着重于确定蛋白水解机制（蛋白水解和ATPase）的各个组件的结构并检验我们的假设。