Conformational dynamics of the ISWI chromatin remodeling enzyme

ISWI 染色质重塑酶的构象动力学

• 批准号: 233780154
• 负责人: Professor Dr. Felix Müller-Planitz
• 金额: --
• 依托单位: Institut für Physiologische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/233780154?language=en
• 关键词: Conformational; dynamics; ISWI; chromatin; remodeling

Nucleosomes densely coat eukaryotic DNA and regulate almost every function of the genome. Highly conserved nucleosome remodeling enzymes control the locations of the nucleosomes, e.g. by ‘sliding’ nucleosomes along DNA. Remodelers of the ISWI family somehow co-opt this sliding activity to even out the spacing between neighboring nucleosomes. Curiously, ISWI assumes a compact, strongly autoinhibited structure when it is not bound to nucleosomes. This ground state is stabilized by multiple autoinhibitory mechanisms. When ISWI binds the nucleosome, stimulatory epitopes on the nucleosome unlock the catalytic potential of the enzyme. We previously proposed that this transformation involves massive conformational rearrangements in the enzyme that involve the ATPase and the C-terminal HAND-SANT-SLIDE (HSS) domains. Our overall aim is to directly visualize the global structural reorganization that occurs when ISWI converts from the autoinhibited to the catalytically active conformation. Importantly, we will expose the mechanistic pathway that underlies this transformation as ISWI encounters the nucleosome, recognizes nucleosomal epitopes and overcomes autoinhibition. We combine quantitative protein crosslinking, mass spectrometry, cryo-electron microscopy (EM), FRET, structural modeling, thermodynamic measurements, substrate analog approaches, mutagenesis, pre-steady-state techniques, and kinetic modeling. The combined approach unveils the choreography between the various biochemical and conformational steps. With the proposed work we will gain unprecedented structural and mechanistic insights how this paradigmatic remodeling machine recognizes its substrate and how the various regulatory elements -much like cogs in an engine- work hand in hand to allow the enzyme to fulfill its biological function. We will gain knowledge how these regulatory elements work, which bears relevance for a better understanding of the enigmatic nucleosome spacing activity, and which will allow us to formulate hypotheses why nature has evolved such an elaborate regulatory framework in the first place. Our study can serve as a blueprint for future dissections of more complex remodeling machines. In-depth mechanistic knowledge and the assays that we will develop could become useful for development of drugs against nucleosome remodelers, a class of enzymes that are drivers of several cancers.

核小体密集地包覆真核生物的DNA并调节基因组的几乎所有功能。高度保守的核小体重塑酶控制核小体的位置，例如通过核小体沿着DNA“滑动”。ISWI家族的重塑者以某种方式利用这种滑动活动来平衡相邻核小体之间的间距。奇怪的是，当ISWI不与核小体结合时，它呈现出一种紧凑的、强烈的自抑制结构。这种基态通过多种自抑制机制来稳定。当ISWI结合核小体时，核小体上的刺激性表位释放酶的催化潜力。我们以前提出，这种转变涉及大量的构象重排的酶，涉及ATP酶和C-末端的手SANT-SLIDE（HSS）域。我们的总体目标是直接可视化的全球结构重组时发生的ISWI从自抑制转化为催化活性构象。重要的是，我们将揭示ISWI遇到核小体，识别核小体表位并克服自抑制的这种转化的机制途径。我们结合了联合收割机定量蛋白质交联，质谱，冷冻电子显微镜（EM），FRET，结构建模，热力学测量，底物模拟方法，诱变，预稳态技术和动力学建模。结合的方法揭示了各种生化和构象步骤之间的编排。通过拟议的工作，我们将获得前所未有的结构和机制见解，这种范式重塑机器如何识别其底物，以及各种调控元件如何-就像发动机中的齿轮-携手工作，让酶实现其生物学功能。我们将获得这些调控元件如何工作的知识，这与更好地理解神秘的核小体间隔活动有关，这将使我们能够提出假设，为什么自然界首先进化出如此复杂的调控框架。我们的研究可以作为未来解剖更复杂的重塑机器的蓝图。深入的机制知识和我们将开发的检测方法可能有助于开发针对核小体重塑的药物，核小体重塑是一类酶，是几种癌症的驱动因素。