Disorganization of the histone core promotes organization of heterochromatin into phase-separated droplets, bioRxiv, 2018-11-19

AbstractThe heterochromatin protein HP1 is proposed to enable chromatin compaction via liquid droplet formation. Yet, a connection between phase separation and chromatin compaction has not been experimentally demonstrated. More fundamentally, how HP1 action at the level of a single nucleosome drives chromatin compaction remains poorly understood. Here we directly demonstrate that the S. pombe HP1 protein, Swi6, compacts arrays of multiple nucleosomes into phase-separated droplets. Using hydrogen-deuterium exchange, NMR, and mass-spectrometry, we further find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a mononucleosome. Restraining these dynamics via site-specific disulfide bonds impairs the compaction of nucleosome arrays into phase-separated droplets. Our results indicate that chromatin compaction and phase separation can be highly coupled processes. Further, we find that such coupling is promoted by a counter-intuitive function of Swi6, namely disorganization of the octamer core. Phase separation is canonically mediated by weak and dynamic multivalent interactions. We propose that dynamic exposure of buried histone residues increases opportunities for multivalent interactions between nucleosomes, thereby coupling chromatin compaction to phase separation. We anticipate that this new model for chromatin organization may more generally explain the formation of highly compacted chromatin assemblies beyond heterochromatin.

biorxiv biophysics 0-100-users 2018

The Dynamic Conformational Landscapes of the Protein Methyltransferase SETD8, bioRxiv, 2018-10-13

Elucidating conformational heterogeneity of proteins is essential for understanding protein functions and developing exogenous ligands for chemical perturbation. While structural biology methods can provide atomic details of static protein structures, these approaches cannot in general resolve less populated, functionally relevant conformations and uncover conformational kinetics. Here we demonstrate a new paradigm for illuminating dynamic conformational landscapes of target proteins. SETD8 (Pr-SET7SET8KMT5A) is a biologically relevant protein lysine methyltransferase for in vivo monomethylation of histone H4 lysine 20 and nonhistone targets. Utilizing covalent chemical inhibitors and depleting native ligands to trap hidden high-energy conformational states, we obtained diverse novel X-ray structures of SETD8. These structures were used to seed massively distributed molecular simulations that generated six milliseconds of trajectory data of SETD8 in the presence or absence of its cofactor. We used an automated machine learning approach to reveal slow conformational motions and thus distinct conformational states of SETD8, and validated the resulting dynamic conformational landscapes with multiple biophysical methods. The resulting models provide unprecedented mechanistic insight into how protein dynamics plays a role in SAM binding and thus catalysis, and how this function can be modulated by diverse cancer-associated mutants. These findings set up the foundation for revealing enzymatic mechanisms and developing inhibitors in the context of conformational landscapes of target proteins.

biorxiv biophysics 200-500-users 2018

 

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