SUMMARY
Histone H3 lysine 79 (H3K79) methylation is enriched on actively transcribed genes, and its misregulation is a hallmark of leukemia. Methylation of H3K79, which resides on the structured disk face of the nucleosome, is mediated by the Dot1L methyltransferase. Dot1L activity is part of a trans-histone crosstalk pathway, requiring prior histone H2B ubiquitylation of lysine 120 (H2BK120ub) for optimal activity. However, the molecular details describing both how Dot1L binds to the nucleosome and why Dot1L is activated by H2BK120 ubiquitylation are unknown. Here, we present the cryoelectron microscopy (cryo-EM) structure of Dot1L bound to a nucleosome reconstituted with site-specifically ubiquitylated H2BK120. The structure reveals that Dot1L engages the nucleosome acidic patch using a variant arginine anchor and occupies a conformation poised for methylation. In this conformation, Dot1L and ubiquitin interact directly through complementary hydrophobic surfaces. This study establishes a path to better understand Dot1L function in normal and leukemia cells.
Organophosphate poisoning can occur from exposure to agricultural pesticides or chemical weapons. This exposure inhibits acetylcholinesterase resulting in increased acetylcholine levels within the synaptic cleft causing loss of muscle control, seizures, and death. Mitigating the effects of organophosphates in our bodies is critical and yet an unsolved challenge. Here, we present a computational strategy that integrates structure mining and modeling approaches, using which we identify novel candidates capable of interacting with a serine hydrolase probe (with equilibrium binding constants ranging from 4 to 120 μM). One candidate Smu. 1393c catalyzes the hydrolysis of the organophosphate omethoate (kcat/Km of (2.0 ± 1.3) × 10−1 M−1s−1) and paraoxon (kcat/Km of (4.6 ± 0.8) × 103 M−1s−1), V- and G-agent analogs respectively. In addition, Smu. 1393c protects acetylcholinesterase activity from being inhibited by two organophosphate simulants. We demonstrate that the utilized approach is an efficient and highly-extendable framework for the development of prophylactic therapeutics against organophosphate poisoning and other important targets. Our findings further suggest currently unknown molecular evolutionary rules governing natural diversity of the protein universe, which make it capable of recognizing previously unseen ligands.
Scientific Reports 6: Article number: 37175; published online: 15 November 2016; updated: 17 February 2017 The Acknowledgements section in this Article is incomplete. “The authors acknowledge David Mowrey, Marino Convertino, and Onur Dagliyan for their assistance in logic discussions. This project is funded by DTRA grant HDTRA-1-12-C-0074.
The original version of this Article contained errors caused by a mistake in the analysis of the fluorescent polarization experimental data. This mistake did not affect the identification of bioscavengers of organophosphates.As a result, in the Abstract, "Here, we present a computational strategy that integrates structure mining and modeling approaches, using which we identify novel candidates capable of interacting with a serine hydrolase probe (with equilibrium binding constants ranging from 4 to 120 μ M)". now reads:"Here, we present a computational strategy that integrates structure mining and modeling approaches, using which we identify novel candidates capable of interacting with a serine hydrolase probe (with equilibrium binding constants ranging from 20 to 120 μ M)".In the Introduction section, "The results from a fluorescence polarization study indicate that all three candidates present OP binding interactions (with equilibrium binding constants ranging from 4 to 120 μ M)". now reads:"The results from a fluorescence polarization study indicate that all three candidates present OP binding interactions (with equilibrium binding constants ranging from 20 to 120 μ M)".In the Results section under subheading 'OP binding to top candidates' , "To quantify OP binding and check for non-covalent interactions, we use fluorescent polarization experiments utilizing the TFP-probe as substrate ( Fig. 3b and Supplementarty Fig. 5). We find that Smu. 1393c has low micromolar binding (K d = 4.17 ± 0.08 μ M). Though candidates' antigen 85-A and phosphoribosyl isomerase show no covalent binding, there are non-covalent binding interactions with K d 's of 20.9 ± 1.6 μ M and 120 ± 10 μ M, respectively. All three candidates display binding interactions that validate the predictions from our computational workflow". now reads:
OPEN
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