Richard J. Hopkinson

PIP2-Binding Site in Kir Channels: Definition by Multiscale Biomolecular Simulations

Phosphatidylinositol bisphosphate (PIP2) is an activator of mammalian inwardly rectifying potassium (Kir) channels. Multiscale simulations, via a sequential combination of coarse-grained and atomistic molecular dynamics, enabled exploration of the interactions of PIP2 molecules within the inner leaflet of a lipid bilayer membrane with possible binding sites on Kir channels. Three Kir channel structures were investigated: X-ray structures of KirBac1.1 and of a Kir3.1−KirBac1.3 chimera and a homology model of Kir6.2. Coarse-grained simulations of the Kir channels in PIP2-containing lipid bilayers identified the PIP2-binding site on each channel. These models of the PIP2−channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. All three channels were revealed to contain a conserved binding site at the N-terminal end of the slide (M0) helix, at the interface between adjacent subunits of the channel. This binding site agrees with mutagenesis data and is in the proximity of the site occupied by a detergent molecule in the Kir chimera channel crystal. Polar contacts in the coarse-grained simulations corresponded to long-lived electrostatic and H-bonding interactions between the channel and PIP2 in the atomistic simulations, enabling identification of key side chains.

Recent Progress in Histone Demethylase Inhibitors

There is increasing interest in targeting histone N-methyl-lysine demethylases (KDMs) with small molecules both for the generation of probes for target exploration and for therapeutic purposes. Here we update on previous reviews on the inhibition of the lysine-specific demethylases (LSDs or KDM1s) and JmjC families of N-methyl-lysine demethylases (JmjC KDMs, KDM2-7), focusing on the academic and patent literature from 2014 to date. We also highlight recent biochemical, biological, and structural studies which are relevant to KDM inhibitor development.

Plant Growth Regulator Daminozide Is a Selective Inhibitor of Human KDM2/7 Histone Demethylases

The JmjC oxygenases catalyze the N-demethylation of N(ε)-methyl lysine residues in histones and are current therapeutic targets. A set of human 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.

Human UTY(KDM6C) Is a Male-specific Nϵ-Methyl Lysyl Demethylase

Background: UTY(KDM6C) has been reported previously to be inactive as a histone demethylase. Results: Crystallography reveals that the fold of the UTY(KDM6C) catalytic domain is highly conserved with those of KDM6A/B. UTY(KDM6C) catalyzes demethylation of Nϵ-methylated lysine histone peptides at Lys27. Conclusion: UTY(KDM6C) is a lysine demethylase that shows high structural similarity with KDM6A/B. Significance: UTY(KDM6C) is a functional Nϵ-methyl lysine demethylase. The Jumonji C lysine demethylases (KDMs) are 2-oxoglutarate- and Fe(II)-dependent oxygenases. KDM6A (UTX) and KDM6B (JMJD3) are KDM6 subfamily members that catalyze demethylation of Nϵ-methylated histone 3 lysine 27 (H3K27), a mark important for transcriptional repression. Despite reports stating that UTY(KDM6C) is inactive as a KDM, we demonstrate by biochemical studies, employing MS and NMR, that UTY(KDM6C) is an active KDM. Crystallographic analyses reveal that the UTY(KDM6C) active site is highly conserved with those of KDM6B and KDM6A. UTY(KDM6C) catalyzes demethylation of H3K27 peptides in vitro, analogously to KDM6B and KDM6A, but with reduced activity, due to point substitutions involved in substrate binding. The results expand the set of human KDMs and will be of use in developing selective KDM inhibitors.

5-Carboxy-8-hydroxyquinoline is a Broad Spectrum 2-Oxoglutarate Oxygenase Inhibitor which Causes Iron Translocation.

2-Oxoglutarate and iron dependent oxygenases are therapeutic targets for human diseases. Using a representative 2OG oxygenase panel, we compare the inhibitory activities of 5-carboxy-8-hydroxyquinoline (IOX1) and 4-carboxy-8-hydroxyquinoline (4C8HQ) with that of two other commonly used 2OG oxygenase inhibitors, N-oxalylglycine (NOG) and 2,4-pyridinedicarboxylic acid (2,4-PDCA). The results reveal that IOX1 has a broad spectrum of activity, as demonstrated by the inhibition of transcription factor hydroxylases, representatives of all 2OG dependent histone demethylase subfamilies, nucleic acid demethylases and γ-butyrobetaine hydroxylase. Cellular assays show that, unlike NOG and 2,4-PDCA, IOX1 is active against both cytosolic and nuclear 2OG oxygenases without ester derivatisation. Unexpectedly, crystallographic studies on these oxygenases demonstrate that IOX1, but not 4C8HQ, can cause translocation of the active site metal, revealing a rare example of protein ligand-induced metal movement.

Arginine demethylation is catalysed by a subset of JmjC histone lysine demethylases

While the oxygen-dependent reversal of lysine Nɛ-methylation is well established, the existence of bona fide Nω-methylarginine demethylases (RDMs) is controversial. Lysine demethylation, as catalysed by two families of lysine demethylases (the flavin-dependent KDM1 enzymes and the 2-oxoglutarate- and oxygen-dependent JmjC KDMs, respectively), proceeds via oxidation of the N-methyl group, resulting in the release of formaldehyde. Here we report detailed biochemical studies clearly demonstrating that, in purified form, a subset of JmjC KDMs can also act as RDMs, both on histone and non-histone fragments, resulting in formaldehyde release. RDM catalysis is studied using peptides of wild-type sequences known to be arginine-methylated and sequences in which the KDMs methylated target lysine is substituted for a methylated arginine. Notably, the preferred sequence requirements for KDM and RDM activity vary even with the same JmjC enzymes. The demonstration of RDM activity by isolated JmjC enzymes will stimulate efforts to detect biologically relevant RDM activity.

Small-molecule-based inhibition of histone demethylation in cells assessed by quantitative mass spectrometry.

Post-translational modifications on histones are an important mechanism for the regulation of gene expression and are involved in all aspects of cell growth and differentiation, as well as pathological processes including neurodegeneration, autoimmunity, and cancer. A major challenge within the chromatin field is to develop methods for the quantitative analysis of histone modifications. Here we report a mass spectrometry (MS) approach based on ultraperformance liquid chromatography high/low collision switching (UPLC-MS(E)) to monitor histone modifications in cells. This approach is exemplified by the analysis of trimethylated lysine-9 levels in histone H3, following a simple chemical derivatization procedure with d(6)-acetic anhydride. This method was used to study the inhibition of histone demethylases with pyridine-2,4-dicarboxylic acid (PDCA) derivatives in cells. Our results show that the PDCA-dimethyl ester inhibits JMJD2A catalyzed demethylation of lysine-9 on histone H3 in human HEK 293T cells. Demethylase inhibition, as observed by MS analyses, was supported by immunoblotting with modification-specific antibodies. The results demonstrate that PDCA derived small molecules are cell permeable demethylase inhibitors and reveal that quantitative MS is a useful tool for measuring post-translational histone modifications in cells.

Optimal Translational Termination Requires C4 Lysyl Hydroxylation of eRF1

Summary Efficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation.

Investigations on the Oxygen Dependence of a 2-Oxoglutarate Histone Demethylase

Histone N(ϵ)-methyl lysine demethylases are important in epigenetic regulation. KDM4E (histone lysine demethylase 4E) is a representative member of the large Fe(II)/2-oxoglutarate- dependent family of human histone demethylases. In the present study we report kinetic studies on the reaction of KDM4E with O2. Steady-state assays showed that KDM4E has a graded response to O2 over a physiologically relevant range of O2 concentrations. Pre-steady state assays implied that KDM4E reacts slowly with O2 and that there are variations in the reaction kinetics which are dependent on the methylation status of the substrate. The results demonstrate the potential for histone demethylase activity to be regulated by oxygen availability.

Monitoring the Activity of 2‐Oxoglutarate Dependent Histone Demethylases by NMR Spectroscopy: Direct Observation of Formaldehyde

Ferrous iron and 2-oxoglutarate (2OG) dependent oxygenases are a diverse superfamily, with members involved in many important biological processes, including oxygen sensing, epigenetic regulation, and collagen, antibiotic, and fatty acid biosynthesis. 2] The 2OG oxygenase histone demethylase (HDM) subfamily catalyses the demethylation of N-methylated lysine residues in the N-terminal tails of histones. Methylated histone tail lysines are involved in the establishment of different chromatin states, and contribute to both gene silencing and activation. Various HDM subfamilies have been implicated in disease states, with the JMJD2 HDMs being linked to prostate and oesophageal cancers. N-Methyllysine demethylation is proposed to occur via hydroxylation of the N-methylated lysine (with concomitant oxidation of the cosubstrate 2OG, and decarboxylation to give carbon dioxide and succinate), followed by fragmentation of the N-hydroxymethyllysine, to give formaldehyde and the demethylated lysine (Scheme 1). The Fe/2OG-dependent oxygenases can be challenging to study in detail, with three substrates (“prime” substrate, 2OG and oxygen) and at least three products (in the case of the histone demethylases, four: demethylated peptide, succinate, formaldehyde and carbon dioxide). NMR spectroscopy is a potentially useful technique for studying 2OG oxygenase reactions in a single assay mixture. Previous reports regarding quantification of formaldehyde released from biocatalysed reactions or formaldehyde levels in biological systems have been based either on its oxidation to formic acid, or on its derivatisation with reagents, such as dimedone or ampicillin. To our knowledge, formaldehyde detection by NMR spectroscopy in enzyme-catalysed reactions has not been reported. Here, we report the use of NMR spectroscopy for monitoring N-demethylation by JMJD2E (a HDM that is sufficiently active for kinetic studies) by monitoring 2OG conversion to succinate, demethylation of N-trimethyl and N-dimethyllysine residues, and both direct and indirect detection of formaldehyde production in demethylation reactions. Initially, the JMJD2E catalysed demethylation reaction was investigated by monitoring the demethylation of octapeptide fragments of the histone H3 N-terminal tail (residues Ala7 to Lys14, N-methylated at residue Lys9). A peptide length of eight amino acids was selected (Ala-Arg-lys(Me3)-Ser-Thr-GlyGly-Lys) in order to allow sufficient substrate recognition by the enzyme whilst reducing signal overlap in the H NMR spectra. A standardised demethylation reaction protocol was developed, firstly by screening a variety of buffers suitable for H NMR spectroscopy (nondeuterated potassium phosphate and ammonium formate buffers, both at 50 mm, pH 7.5) and then by optimising concentrations of reagents to allow NMR detection. JMJD2E only displayed sufficient activity in ammonium formate buffer, and this was selected for further work. l-Ascorbate has been previously shown to increase activity of some Fe/2OG-dependent oxygenases and was therefore included in the assay mixture. The presence of Fe ions at 100 mm did not cause any noticeable loss of resolution. The demethylation reactions with triand dimethylated peptides (K9me3 and K9me2, respectively) under optimised conditions Scheme 1. Proposed mechanism for JMJD2-catalysed demethylation. Oxidative decarboxylation of 2OG generates a Fe=O species, which reacts with the methylated lysine residue. Hydroxymethyllysine then fragments to give formaldehyde and the demethylated product. The demethylated carbon is highlighted in bold; R = H or CH3.