Mechanisms of Hsp100 chaperones

Hsp100 分子伴侣的机制

• 批准号: 62379277
• 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
• 财政年份: 2008
• 资助国家: 德国
• 起止时间: 2007-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/62379277?language=en
• 关键词: Mechanisms; Hsp100; chaperones

Hsp100 proteins are ring-forming and ATP-fueled machines that remodel and unfold substrate proteins. Hsp100s play crucial roles in bacterial physiology, virulence and stress resistance by linking ATP-dependent threading of substrates to degradation or refolding processes. Substrate selectivities and ATPase activities of Hsp100 chaperones are tightly controlled and loss of such control is deleterious for cells. Accordingly, deregulation of Hsp100s by small molecules is increasingly recognized as novel antibacterial strategy. Hsp100s are typically repressed in the ground state and require binding of adapters and substrates to reach an activated state with high ATPase and threading activity. The modes of Hsp100 activity control are diverse and activated Hsp100s also differ in ATPase activity and threading processivity. Control mechanisms are therefore tailored to the specific functional needs of a given Hsp100 in protein remodeling.We previously revealed several mechanistic aspects relevant to this proposal. First, we determined the basic control mechanisms of the disaggregase ClpB and the unfoldase ClpC, which are activated through binding of partner proteins (Hsp70, MecA) to regulatory M-domains (MDs). A detailed dissection of the ClpB ATP hydrolysis mechanism combined with cryo EM structural analysis suggests that activation induces a sequential mode of ATP hydrolysis, coupled to coordinated substrate threading. The processivity of substrate threading is lower for ClpB as compared to other Hsp100s pointing to a so far unknown mechanism that limits the time length of activation. Second, we showed that the kinase McsB and the antibacterial small molecule Cyclomarin A (CymA) activate ClpC in a MD-independent manner, indicating novel pathways for ClpC activation. Third, we identified ClpG as stand-alone disaggregase, which confers superior heat resistance to bacteria. ClpG itself selectively binds to protein aggregates via a unique N-terminal domain. We plan to further explore the diverse mechanisms of Hsp100 activity control and substrate selection that are underlying Hsp100 functions and concurrently protecting cells from deleterious Hsp100 activities. We will address the following major questions:- How do intra- and interring communications propel a sequential ATP hydrolysis mode upon ClpB activation? Which mechanism confines ClpB threading processivity to maximize disaggregase function? - How do CymA and McsB activate ClpC independent from MDs? What is the mechanistic basis of toxic ClpC-activation by CymA?- What is the structural basis for the unique ability of ClpG to independently and selectively recognize aggregated but not soluble misfoded proteins?With these approaches we are aiming at a comprehensive understanding of the discrete steps controlling Hsp100 activities: substrate targeting, ATPase activation by partners and substrates and the conversion of high ATP rates upon activation into a substrate processing mechanical force.

Hsp100蛋白质是一种环形成和ATP驱动的机器，可以重塑和展开底物蛋白质。Hsp100在细菌生理、毒力和抗逆性中发挥重要作用，它通过将依赖于ATP的底物穿梭与降解或复性过程联系起来。Hsp100的底物选择性和ATPase活性受到严格控制，失去这种控制对细胞是有害的。因此，通过小分子来解除对Hsp100的调控被认为是一种新的抗菌策略。Hsp100通常被抑制在基态，需要适配器和底物的结合才能达到具有高ATPase和线活性的激活状态。Hsp100的活性调控方式多种多样，激活的Hsp100在ATPase活性和线程性上也不同。因此，控制机制是根据给定Hsp100在蛋白质重构中的特定功能需求而定制的。我们先前揭示了与这一提议相关的几个机制方面。首先，我们确定了解聚酶ClpB和解折叠酶ClpC的基本控制机制，这两种酶是通过结合伙伴蛋白(Hsp70，MecA)和调节M结构域(MD)而激活的。结合低温EM结构分析，对ClpB的ATP水解机制的详细剖析表明，激活诱导了一种顺序的ATP水解模式，并与配位的底物穿线相结合。与其他Hsp100相比，ClpB的底物穿线加工能力较低，这表明一种迄今未知的机制限制了激活的时间长度。其次，我们发现MCSB和抗菌小分子环素A(CymA)以非MD依赖的方式激活ClpC，这表明了ClpC激活的新途径。第三，我们确定ClpG是一种独立的解聚酶，它对细菌具有优异的耐热性。ClpG本身通过一个独特的N-末端结构域选择性地与蛋白质聚集体结合。我们计划进一步探索Hsp100活性控制和底物选择的不同机制，这些机制是Hsp100功能的基础，同时保护细胞免受有害的Hsp100活性的影响。我们将解决以下主要问题：-在ClpB激活后，内部和内部通信如何推动顺序的ATP水解模式？哪种机制限制了ClpB的线程处理能力以最大限度地发挥解聚酶功能？-Cyma和MCSB如何独立于MD激活ClpC？CymA激活有毒ClpC的机制是什么？-ClpG具有独立和选择性地识别聚集但不能溶解的错误蛋白质的独特能力的结构基础是什么？通过这些方法，我们旨在全面了解控制Hsp100活性的离散步骤：底物靶向，伙伴和底物激活ATPase，以及激活后高ATP速率转化为底物加工机械力。