Conformational dynamics of the ISWI chromatin remodeling enzyme

核小体密集地包裹着真核生物的DNA，并调节着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，例如通过沿DNA“滑动”核小体。ISWI家族的重塑者以某种方式利用这种滑动活性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，它具有紧密的、强烈的自抑制结构。这种基态是由多种自抑制机制稳定的。当ISWI结合核小体时，核小体上的刺激表位释放酶的催化潜能。我们之前提出，这种转化涉及atp酶和c端HAND-SANT-SLIDE （HSS）结构域的酶的大量构象重排列。我们的总体目标是直接可视化ISWI从自抑制构象转变为催化活性构象时发生的全局结构重组。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身抑制时这种转化的机制途径。我们结合定量蛋白质交联，质谱，低温电子显微镜（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。通过提出的工作，我们将获得前所未有的结构和机制方面的见解，这种典型的重塑机器如何识别其底物，以及各种调节元件（非常像发动机中的齿轮）如何携手合作，使酶实现其生物功能。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，并将使我们能够提出假设，为什么大自然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对开发针对核小体重塑酶的药物有用，核小体重塑酶是几种癌症的驱动因素。

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.