Structure of functionally important dynamic states of the proteasome

描述（由申请人提供）：蛋白酶体是以受调控的方式降解细胞内蛋白质的巨大分子复合物。它们由两个部分组成-19 S调节颗粒（RP）和蛋白水解20 S催化颗粒（CP）。的目的 应用是了解功能相关的蛋白酶体结构动力学。几个实验室的最新发现提供了蛋白酶体的亚纳米分辨率结构。然而，这些美丽的模型是不完整的。第一，基质不存在。在第一个具体目标中， 将确定蛋白质底物如何与蛋白酶体结合。本研究将探讨底物与蛋白酶体三个关键作用位点的相互作用模式：底物结合、延伸的多肽主动转位和折叠结构域解链的收缩。第二，现有的模型是静态的。在第二个具体目标中，将使用各种生物化学方法来捕获底物与蛋白酶体在一组与不同功能状态相关的特定构象状态中的接合。一 广泛接受但未经检验的范例是蛋白质解折叠和移位与蛋白酶体的大规模构象变化相结合。这一观点将通过确定蛋白酶体的结构与特定状态下的底物加工进行测试。第三个具体目标将调查最近的生化观察表明蛋白酶体CP内的同向性变构的结构基础。CP内的通信可能是重要的生化调节，并在确定细胞如何分配CP池之间的各种形式的经典和杂交蛋白酶体。使用两个PI构建本申请的基本原理是，实现本项目的科学目标需要他们持续相互作用，以利用和进一步开发源自每个申请人实验室的方法：新型cryoEM方法，可实现快速高分辨率结构测定，以及新型蛋白酶体底物，可形成稳定和可调的蛋白酶体-底物复合物用于结构研究。拟议的研究将可视化功能相关的蛋白酶体复合物在原子水平的分辨率。蛋白酶体由ATP结合和水解提供能量，并使用由此获得的能量来移动和展开其底物。然而，很少有信息是有关蛋白酶体功能的结构， 动力学这些研究的长期目标是了解蛋白酶体作用的生物力学。这项研究意义重大，因为它是将我们对蛋白酶体的理解从静态对象转变为动态参与者的第一步。

DESCRIPTION (provided by applicant): Proteasomes are giant molecular complexes that degrade intracellular proteins in a regulated manner. They are composed of two subassemblies- a 19S regulatory particle (RP) and a proteolytic 20S catalytic particle (CP). The objective of this application is to understand functionally relevant proteasome structural dynamics. Recent findings from several labs provide subnanometer resolution structures of the proteasome. These beautiful models, however, are incomplete. First, substrate is absent. In the first Specific Aim it will be determined how protein substrates engage the proteasome. This Aim will investigate the mode of interaction between substrate and three key proteasome sites of action: substrate binding, extended polypeptide undergoing active translocation and the constriction against which a folded domain unravels. Second, the present models are static. In the second Specific Aim various biochemical approaches will be used to trap substrate engagement with proteasome in a set of specific conformational states associated with distinct functional states. A widely accepted yet untested paradigm is that protein unfolding and translocation is coupled with large-scale conformational changes of the proteasome. This view will be tested by determining structures of proteasomes with substrate processing in specific states. The third Specific Aim will investigate the structural basis of recent biochemical observations demonstrating homotropic allostery within the proteasome CP. Communication within the CP may be important in biochemical regulation and in determining how the cell allocates the CP pool among various forms of classic and hybrid proteasomes. The rationale for structuring this application with two PIs is that accomplishing the scientific goals of this project requires their continuing interaction to exploit and further develop methods that originated in the labs of each of the applicants: Novel cryoEM methods, which enable rapid high-resolution structure determination, and novel proteasome substrates, which enable formation of stable and tunable proteasome-substrate complexes for structural studies. The proposed studies will visualize functionally relevant proteasome complexes at atomic- level resolution. Proteasomes are fueled by ATP binding and hydrolysis and use the energy so derived to move and unfold their substrates. However, little information is available that relates proteasome function to structural dynamics. The long term goal of these investigations is to understand the biomechanics of proteasome action. The proposed research is significant because it is a first step in transforming our understanding of proteasomes from static objects to dynamic players.