Allosteric mechanisms of AAA+ machines: the case of the central bacterial protease ClpC/ClpP

AAA 机器的变构机制：中心细菌蛋白酶 ClpC/ClpP 的案例

• 批准号: 497321492
• 负责人: Privatdozent Dr. Axel Mogk
• 金额: --
• 依托单位: Zentrum für Molekulare Biologie (ZMBH)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/497321492?language=en
• 关键词: Allosteric; mechanisms; AAA+; machines; case

Hsp100 chaperones are ring-forming ATP-fueled protein quality machines that play crucial roles in bacterial physiology and virulence. They use mechanical work to thread substrates through their central channel, leading to disassembly or unfolding of cellular targets. Many Hsp100 proteins (e.g. ClpC) associate with peptidases (e.g. ClpP) to form bacterial proteasomes. The degradation activities of proteases are potentially deleterious; substrate selection and ATPase activities of Hsp100 proteins are therefore tightly controlled. This is achieved by specific partner proteins (adaptors) that provide substrate specificity and strongly increase Hsp100 ATPase activity upon substrate transfer. Persistent, adaptor-independent activation of Hsp100 proteins is highly toxic to cells and therefore represents a useful anti-bacterial strategy. There are multiple conundrums related to the mechanism of operation of Hsp100 proteins. Here we propose to use ClpC/ClpP, the central protease of the important pathogen Staphylococcus aureus, as a model that will allow us to tackle many of these open questions. ClpC is composed of two ATPase domains that form two distinct rings in the hexameric assembly and additional domains that mediate binding to diverse adaptors. It combines with the heptameric peptidase ClpP to form an effective ATP-driven protein degradation machine. We would like to understand: How is the activity of the machine regulated by various binding effectors? How does the hexameric ClpC cooperate with the heptameric peptidase ClpP during function? And how is ATP hydrolysis coupled to substrate transport through the protein’s pore? We plan to tackle these key questions by combining structural, biochemical and single-molecule protein dynamics studies, which rely on the strong complementary expertise of the two participating groups. We base our plans on our vast experience in the Hsp100 field and on the expertise of Gilad Haran’s lab in applying single-molecule FRET (smFRET) spectroscopy to study the dynamics of complex molecular machines. In particular, we will (i) define the structural basis of the specific regulatory circuits controlling ClpC function by a combination of cryo-EM structure determination of Hsp100 hexamers in diverse activity states with smFRET studies of machine dynamics. We will further (ii) study machine mechanics by directly observing substrate threading through the central pore in real time, and exploring (potentially rotational) motions at the symmetry mismatched interface of the ClpC hexamer and the ClpP heptamer. The project will culminate with (iii) a search for small-molecule activators of ClpC that can serve as potential novel antibiotics, thus bringing the biophysical-biochemical knowledge acquired here close to medical application.

Hsp100伴侣蛋白是由atp驱动的环状蛋白质量机器，在细菌生理和毒力中起着至关重要的作用。它们使用机械工作将底物穿过其中央通道，导致细胞靶标的拆卸或展开。许多Hsp100蛋白（如ClpC）与肽酶（如ClpP）结合形成细菌蛋白酶体。蛋白酶的降解活性具有潜在的危害性；因此，Hsp100蛋白的底物选择和atp酶活性受到严格控制。这是通过提供底物特异性并在底物转移时强烈增加Hsp100 atp酶活性的特定伴侣蛋白（适配器）实现的。Hsp100蛋白的持续、不依赖于接头的激活对细胞具有高毒性，因此代表了一种有用的抗菌策略。关于Hsp100蛋白的运作机制，存在着多个难题。在这里，我们建议使用重要病原体金黄色葡萄球菌的中心蛋白酶ClpC/ClpP作为模型，这将使我们能够解决许多悬而未决的问题。ClpC由两个atp酶结构域组成，它们在六聚体组装中形成两个不同的环，以及介导与不同接头结合的附加结构域。它与七聚体肽酶ClpP结合形成一个有效的atp驱动的蛋白质降解机器。我们想了解：机器的活动是如何被各种结合效应器调节的？六聚体ClpC在功能过程中如何与七聚体肽酶ClpP合作？ATP水解是如何通过蛋白质孔与底物运输耦合的？我们计划通过结合结构、生化和单分子蛋白质动力学研究来解决这些关键问题，这些研究依赖于两个参与小组强大的互补专业知识。我们的计划基于我们在Hsp100领域的丰富经验和Gilad Haran实验室在应用单分子FRET （smFRET）光谱研究复杂分子机器动力学方面的专业知识。特别是，我们将(i)通过结合Hsp100六聚体在不同活性状态下的低温电镜结构测定和机器动力学的smFRET研究来定义控制ClpC功能的特定调节电路的结构基础。我们将进一步（ii）研究机器力学，直接观察基材穿过中心孔的实时情况，并探索ClpC六聚体和ClpP七聚体对称不匹配界面的运动（可能是旋转）。该项目最终将以（iii）寻找ClpC的小分子激活剂作为潜在的新型抗生素，从而使这里获得的生物物理生化知识接近医学应用。