Allosteric Mechanism of Hsp70 Molecular Chaperones

描述（由申请人提供）：普遍存在的Hsp 70分子伴侣家族利用看似简单的结构域间变构机制来实现惊人的广泛的关键细胞功能。热休克蛋白70促进新合成的蛋白质的折叠，通过拯救错误折叠的蛋白质或将其引导至降解机构来保护细胞免受在应激条件下可能发生的损伤，帮助蛋白质跨膜移位，并促进大分子复合物的组装和拆卸。所有这些功能都依赖于Hsp 70结合蛋白质底物的未折叠区域的能力，以及在ATP的变构结合后释放其底物的能力。Hsp 70由于其抗凋亡和致瘤活性而成为癌症治疗的潜在靶点， 并且由于它们能够拯救易于聚集的蛋白质而作为与蛋白质错误折叠相关的疾病（囊性纤维化、神经变性疾病）的靶标。我们的工作使用E. coli Hsp 70，DnaK作为Hsp 70变构的基本机制的一个范例，显示了变构信号是如何从N-末端结构域的核苷酸结合位点传递到结构域间的连接体的，并且我们对这如何导致C-末端底物结合结构域（SBD）的主要重组和伴随的底物亲和力降低有了诱人的提示。在目标1中，我们将建立在我们最近的进展和映射的变构信号传输DnaK到SBD，使用新的核磁共振方法的组合大蛋白质，和互补的生物物理方法，如脉冲电子自旋共振。我们将研究如何共同分子伴侣相互作用重塑DnaK变构景观，以及我们新发现的极端DnaK C-末端的作用可能会影响其与底物的相互作用。在目标2中，通过我们正在进行的DnaK工作收集的方法和知识将应用于四种人类Hsp 70，细胞质Hsc 70（组成型）和HspA 1（诱导型），ER驻留BiP和线粒体mtHsp 70。这些分子伴侣机器共享一个共同的机制，但其特征在于大量的序列多样化和随之而来的底物偏好和共分子伴侣的差异。在目标3中，我们将从结构和生物药理学上表征合作者提供给我们的Hsp 70小分子调节剂，这些合作者正在探索Hsp 70作为药物靶点。我们将通过NMR确定候选小分子与Hsp 70的相互作用模式，寻找对特定Hsp 70的选择性的证据以及如何增强它，并提供反馈以改善调节剂的特性。总的来说，这项研究将有助于我们对变构的基本理解，同时为不同Hsp 70家族成员的功能和专业化提供见解。这些见解对于Hsp 70作为药物靶标的最佳使用至关重要。 公共卫生相关性：热休克蛋白70分子伴侣在正常生理条件下发挥重要作用，保护细胞免受热休克等应激。Hsp 70与包括癌症和蛋白质错误折叠疾病（如囊性纤维化、阿尔茨海默病和亨廷顿病）在内的几种毁灭性疾病有关，导致它们成为可能的治疗靶点。本研究将为深入了解Hsp 70的基本作用机制和Hsp 70亚家族的功能多样性提供理论依据，从而为Hsp 70作为药物靶点的应用提供理论基础。

DESCRIPTION (provided by applicant): The ubiquitous Hsp70 family of molecular chaperones utilizes a deceptively simple mechanism of interdomain allostery to accomplish a stunningly broad array of critical cellular functions. Hsp70s facilitate folding of newly synthesizd proteins, protect cells from damage that can occur under stress conditions by either rescuing misfolded proteins or directing them to degradation machinery, assist protein translocation across membranes, and promote assembly and disassembly of macromolecular complexes. All of these functions rely on the ability of Hsp70s to bind unfolded regions of a protein substrate, and to release their substrates upon allosteric binding of ATP. Hsp70s have emerged as potential targets for cancer therapeutics because of their anti-apoptotic and tumorigenic activity, and as targets for diseases associated with protein misfolding (cystic fibrosis, neurodegenerative diseases) because of their ability to rescue aggregation-prone proteins. Our work using the E. coli Hsp70, DnaK, as a paradigm for the fundamental mechanism of Hsp70 allostery has shown how allosteric signals are communicated from the nucleotide-binding site of the N-terminal domain to the interdomain linker, and we have tantalizing hints about how this then causes major reorganization and concomitant reduced substrate affinity in the C-terminal substrate-binding domain (SBD). In Aim 1, we will build on our recent progress and map the allosteric signal transmission in DnaK into the SBD, using a combination of novel nuclear magnetic resonance approaches for large proteins, and complementary biophysical methods such as pulsed electron spin resonance. We will study how co-chaperone interactions remodel the DnaK allosteric landscape, and how our newly discovered role for the extreme C-terminus of DnaK may influence its interactions with substrates. In Aim 2, the methods and knowledge gathered through our ongoing work on DnaK will be applied to four human Hsp70s, the cytoplasmic Hsc70 (constitutive) and HspA1 (inducible), the ER-resident BiP, and the mitochondrial mtHsp70. These chaperone machines share a common mechanism but are characterized by substantial sequence diversification and consequent differences in substrate preferences and in co-chaperone partners. In Aim 3, we will structurally and biophysically characterize small molecule modulators of Hsp70s provided to us by collaborators who are exploring Hsp70s as drug targets. We will determine by NMR the mode of interaction of candidate small molecules with Hsp70s, look for evidence of selectivity for particular Hsp70s and how it may be enhanced, and provide feedback to improve characteristics of modulators. Overall, the research proposed will contribute to our fundamental understanding of allostery, while providing insights into function and specialization of different Hsp70 family members. These insights are critical for optimal use of Hsp70s as drug targets. PUBLIC HEALTH RELEVANCE: Hsp70 molecular chaperones play key roles under normal physiological conditions and protect cells against stresses such as heat shock. Hsp70s have been implicated in several devastating diseases including cancers and protein misfolding diseases, such as cystic fibrosis, Alzheimer's, and Huntington's, leading to their emergence as possible therapeutic targets. This research will provide much needed knowledge about basic mechanisms of Hsp70s and functional diversity among Hsp70 subfamilies and thus will greatly facilitate their use as drug targets.