Conformational dynamics of the ISWI chromatin remodeling enzyme

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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.