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Catalysis by the JmjC histone demethylase KDM4A integrates substrate dynamics, correlated motions Cite this: Chem. Sci., 2020, 11, 9950 † All publication charges for this article and molecular orbital control have been paid for by the Royal Society a a b of Chemistry Rajeev Ramanan, Shobhit S. Chaturvedi, Nicolai Lehnert, Christopher J. Schofield, c Tatyana G. Karabencheva-Christova *a and Christo Z. Christov *a
3 The N -methyl lysine status of histones is important in the regulation of eukaryotic transcription. The Fe(II) and 2-oxoglutarate (2OG) -dependent JmjC domain enzymes are the largest family of histone N3-methyl lysine demethylases (KDMs). The human KDM4 subfamily of JmjC KDMs is linked with multiple cancers and some of its members are medicinal chemistry targets. We describe the use of combined molecular dynamics (MD) and Quantum Mechanical/Molecular Mechanical (QM/MM) methods to study the mechanism of KDM4A, which catalyzes demethylation of both tri- and di-methylated forms of histone
Creative Commons Attribution-NonCommercial 3.0 Unported Licence. H3 at K9 and K36. The results show that the oxygen activation at the active site of KDM4A is optimized towards the generation of the reactive Fe(IV)-oxo intermediate. Factors including the substrate binding mode, correlated motions of the protein and histone substrates, and molecular orbital control synergistically contribute to the reactivity of the Fe(IV)-oxo intermediate. In silico substitutions were performed to investigate the roles of residues (Lys241, Tyr177, and Asn290) in substrate orientation. The Lys241Ala substitution abolishes activity due to altered substrate orientation consistent with reported experimental studies. Calculations with a macrocyclic peptide substrate analogue reveal that induced conformational changes/correlated motions in KDM4A are sequence-specific in a manner that influences substrate binding affinity. Second sphere residues, such as Ser288 and Thr289, may This article is licensed under a contribute to KDM4A catalysis by correlated motions with active site residues. Residues that stabilize key Received 6th July 2020 intermediates, and which are predicted to be involved in correlated motions with other residues in the Accepted 4th September 2020 second sphere and beyond, are shown to be different in KDM4A compared to those in another JmjC DOI: 10.1039/d0sc03713c KDM (PHF8), which acts on H3K9 di- and mono-methylated forms, suggesting that allosteric type Open Access Article. Published on 04 September 2020. Downloaded 10/2/2021 10:32:33 PM. rsc.li/chemical-science inhibition is of interest from the perspective of developing selective JmjC KDM inhibitors.
1. Introduction methylation states and histone substrate sequence selectiv- ities.3,4 The KDM1 subfamily, or lysine-specic demethylases Epigenetic processes regulate eukaryotic development and are (LSDs), are avin-dependent enzymes that catalyze demethyla- involved in diseases, including cancer, metabolic syndromes, tion of di- and mono-methylated forms of lysine-4 in histone 3 and brain disorders.1 Post-translational modications of histone tails in nucleosomes, including via reversible lysine- and arginine N-methylation and demethylation, are crucial mechanisms of transcriptional regulation (Scheme 1).2,3 3 There are nine groups of identied human N -methyl lysine 3 demethylases (KDMs) (KDM1-9) which act on different N -
aDepartment of Chemistry, Michigan Technological University, Houghton, Michigan 49931, USA. E-mail: [email protected]; [email protected] bDepartment of Chemistry, University of Michigan, Ann Arbor, MI, 48019, USA cThe Chemistry Research Laboratory, University of Oxford, Manseld Road, OX1 5JJ, UK † Electronic supplementary information (ESI) available: Potential energy proles, cross-correlation plots, orbital pictures, Mulliken spin and charges and Cartesian 3 coordinates of the QM zone in QM/MM calculations are provided. See DOI: Scheme 1 N -Lysine methylation and demethylation of histone H3 10.1039/d0sc03713c play important roles in the regulation of transcription.
9950 | Chem. Sci.,2020,11,9950–9961 This journal is © The Royal Society of Chemistry 2020 View Article Online Edge Article Chemical Science
a 3 b–d (H3K4me2/me1) substrates.3 KDM2-9 are non-heme Fe(II) – and demethylation of all three N -lysine methylation states.3 – 2-oxoglutarate (2OG) dependent Jumonji C (JmjC) domain- KDM2-9 employ an Fe(II) cofactor, and 2OG and O2 as cosub- containing oxygenases that are capable of catalyzing strates. The chemical basis of the underlying differences in Creative Commons Attribution-NonCommercial 3.0 Unported Licence. This article is licensed under a Open Access Article. Published on 04 September 2020. Downloaded 10/2/2021 10:32:33 PM.
Fig. 1 Outline of catalytic cycle for the JmjC KDMs.
This journal is © The Royal Society of Chemistry 2020 Chem. Sci.,2020,11, 9950–9961 | 9951 View Article Online Chemical Science Edge Article
selectivity between the JmjC KDM subfamilies is poorly We report studies aimed at testing the proposal that, understood. although the catalytic domains of KDMs appear to employ the The human KDM4 subfamily comprises ve multi-domain same mechanisms and share substantial structural similarity, enzymes (KDM4A–E),5 which catalyze demethylation of the tri- catalytically important interactions unique to specic sets of and di-methylated lysine residue K9 in histone H3 N-terminal KDMs exist. We envisaged that in addition to enzyme-specic tails (H3K9me3/me2).5,6 KDM4A–C (which have >50% rst-sphere catalytic residues, there would be a broader sequence identity over their core JmjC domain, JmjN, two plant enzyme-specic network of correlated motions and interactions homeodomains (PHD) and two hybrid Tudor domains)5 also between residues directly involved in catalysis and second catalyze H3K36me3/me2 demethylation, but KDM4D/E do not.7 sphere and beyond residues. We carried out molecular Demethylation of H3K9me3 by the JmjC KDM4s facilitates an dynamics (MD) and quantum mechanical/molecular mechan- open chromatin state, contributing to transcriptional activa- ical (QM/MM) calculations on the roles of structural dynamics tion.8 Multiple common cancers, including breast,9 prostate,10 and long-range correlated motions in KDM4A with two types of and lung11 cancer are associated with KDM4A gene over- substrate – histone H3 peptides and a tight binding unnatural expression.10c,11c,12–14 Thus, KDM4 and other JmjC KDMs are macrocyclic peptide substrate analogue.21c These results are being pursued as targets for anti-cancer therapeutics.15 further compared to those for KDM7B (PHF8) from previous The JmjC KDM catalytic cycle is proposed to follow the work, and interesting differences are analyzed and related back consensus for 2OG oxygenases, as established by spectro- to the substrate specicity of these enzymes. scopic,16 kinetic,17 and computational studies,18 (Fig. 1). In the resting enzyme, the Fe(II) is octahedrally-coordinated by His- 2. Methodology 188, Glu-190 and His-276 (for KDM4A) and two to three water molecules. 2OG binding to A generate B (Fig. 1), is followed by Crystal structures of the catalytic domain of KDM4A bound to that of the substrate, then dioxygen. Work with multiple non- the H3(7–12)K9me3 natural substrate (PDB ID: 2OQ6)21a or to II Creative Commons Attribution-NonCommercial 3.0 Unported Licence. heme Fe( ) enzymes reveals that the presence of a substrate is a cyclic peptide substrate analogue/inhibitor (PDB ID: 5LY1) necessary for productive oxygen binding and reaction to form were used.21c A crystal structure of PHF8 complexed with an a ferric-superoxo complex (RC1, Fig. 1). Oxidative decarboxyl- H3(1–14)K4me3K9me2 substrate fragment (PDB ID: 3KV4)21b
ation of 2OG results in CO2 and an Fe-linked succinyl peroxide was treated similarly to the KDM4 structures. Amber16 was intermediate IM1 (Fig. 1). Subsequent O–O homolysis gives used for the MD simulations.25 ChemShell suite of programs a ferryl intermediate (IM2, Fig. 1) (S ¼ 2, M ¼ 5), which abstracts was used for QM/MM calculations.26,27 The QM calculations a hydrogen from the substrate to give IM3; a subsequent were performed with Turbomole,28 and MM with DL_POLY rebound (IM3 to PD) provides a hemiaminal, which eliminates soware.29 All geometries were optimized using the B3LYP30 formaldehyde to provide the demethylated product.19 Water density functional theory with the def2-SVP (split valence
This article is licensed under a ligation regenerates the resting state. The nal step likely polarization) basis set (B1) which can reproduce accurate proceeds non-enzymatically, at least for some Fe(II)/2OG barriers and electronic structure for Fe metal containing sys- oxygenases.19 tems.23a,31,32 Details are provided in ESI† of the article. The JmjC KDMs have a characteristic distorted double Open Access Article. Published on 04 September 2020. Downloaded 10/2/2021 10:32:33 PM. stranded beta-helix (DSBH) core fold, as conserved in 2OG 3. Results and discussion oxygenases.20 Elements surrounding the core DSBH fold are subfamily characteristic and are involved in substrate binding.21 3.1 Conformational exibility and correlated motions in the – One end of the DSBH supports the active site containing the reactant complex: Fe(III)-superoxo complex with H3(7 12) single Fe ion. The KDM4 catalytic domains also contain a Zn K9me3 binding site which is structurally important.21a,c Single substi- To date the proposed reactive intermediates in KDM4 catalysis tutions of residues, not in the immediate vicinity of the Fe- have not been characterized experimentally, hence we carried
center, can impact substantially on KDM4 activity, e.g. second out studies on the KDM4A Fe(III)–O2$2OG$H3(7–12)K9me3 sphere residue substitutions in KDM4A (e.g. K241A, ST288- complex (RC1 in Fig. 1), initially by performing an MD simu- 289TV/NV/GG) can ablate activity.22b Similarly, the ST288- lation of 1 ms (Fig. 3). The KDM4A active site orients both 2OG m 289AI KDM4A substitution alters binding of both di- and tri- and O2 for apparently productive reaction during the entire 1 s methylated H3-K9me2/3 substrates.22 MD simulation. Distance plots reecting the relative orientation
Computational studies on KDMs have been focused on the of the O2 derived atoms and 2OG (Fig. 3) and between the tri- 3 conformational dynamics and the reaction mechanism in the methylated N -lysine and the proximal oxygen from O2 (Op–N) KDM7 (ref. 23) enzymes and dioxygen binding in KDM4A.24 show only small variations, implying a stable enzyme–substrate There is little understanding of the key interactions involved in complex. The 2OG and the substrate orientations are stabilized directing the reaction mechanism of KDM4A and how these by hydrogen bonds and other interactions (Fig. 2 and S2c in differ between KDMs in different subfamilies. Knowledge of ESI†). The C5 oxygens of 2OG (O3 and O4 in Fig. 3) are stabilized long-range correlated motions important in catalysis is not only by polar interactions with Tyr132 and Lys206. Asn198 forms of basic mechanistic interest, but by identifying enzyme specic a stable hydrogen bond with O1 oxygen of 2OG. The results thus processes it may help enable the design of selective inhibitors. imply that Tyr132, Lys206, and Asn198, stabilize productive binding of 2OG to Fe, as implied by the crystal structures.21a,c
9952 | Chem. Sci.,2020,11,9950–9961 This journal is © The Royal Society of Chemistry 2020 View Article Online Edge Article Chemical Science
Fig. 2 Views of the KDM4A$Fe(III)$O2-substrate (H3(7–12)K9me3) complex. (a) Geometry of the optimized ferric-superoxo [Fe(III)–O2] complex for KDM4A. (b) The quantum mechanical region used in calculations. Wiggly lines define the boundary of the QM/MM partition. The views were
derived from a modeled structure of Fe(III)–O2 complexed with H3(7–12)K9me3 (based PDB: 2OQ6) as described in Methods. Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
Fig. 3 Distances between the dioxygen derived proximal oxygen (Op) to the substrate N-methyl N (Op–N) (in red) and of the distal oxygen (Od)
This article is licensed under a to the 2OG co-substrate (C2–Od) (in black) during the 1 ms simulation. The x-axis denotes time in ns, and the y-axis denotes distance in A.˚ Atom labels are shown in the inset.
Open Access Article. Published on 04 September 2020. Downloaded 10/2/2021 10:32:33 PM. Due to correlated motions, second sphere residues not two of the three Fe-coordinating residues (see Fig. 1); Ser-288 and directly coordinated to the metal ion can inuence catalysis.33 To Thr-289 are second sphere residues located on b15, which is one investigate roles of correlated motions in KDM4A catalysis, we of the 8 DSBH b-strands (secondary structure elements of carried out dynamic cross-correlation analyses (DCCA)34 for the KDM4A are given in Fig. S1 of ESI†). The experimentally KDM4A Fe(III)-superoxo$H3(7–12)K9me3 complex (RC1 in observed ablation of KDM4A activity with the ST288-289TV, NV, Fig. 2). The results (Fig. 4) reveal strong correlated motions or GG variants and altered binding specicity for both dime- between residues 258–300 and 188–213. His-188 and Glu-190 are thylated (H3-K9me2) and trimethylated (H3-K9me3) substrates
Fig. 4 Motions in the Fe(III)–O2$2OG H3(7–12)K9me3 complex. (a) Dynamic cross-correlation diagram with important correlations circled. Residues 158–170 belong to a5 and b6; 227–234 belong to a7. (b) Principal component analysis showing the direction of motions yellow to blue.
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by ST288-289AI may thus, at least in part, be due to alterations in shi is likely to the p* orbitals based on the angle of HAT correlated motions involving residues 288–289 along with other trajectory that emanate from a nonlinear positioning of possible alterations in the electrostatic eld.22b Principal substrate with respect to Fe–O bond.18h,36,37 The exchange Component Analysis (PCA) (Fig. 4b) shows the directions of enhanced reactivity makes the s pathway preferred over the p motion of the protein and the relative exibility of specic pathway.36,37 protein regions during the simulation.34 Backbone residues MD simulations of the KDM4A Fe(IV)-oxo intermediate (IM2) located at a5, b6 (158 to 170) and a7 (227 to 234) show relatively show that Tyr132 and Lys241 are positioned to make hydrogen larger movements compared to other regions of the protein. bonds with the C4 oxygens of succinate, and Asn198 is oriented to make a hydrogen bond with the non-coordinating C1 oxygen (O2) of the succinate (Fig. S6†). With KDM4A, Glu190 is posi- 3.2 Interactions and correlated motions in the Fe(IV)]O tioned to make a hydrogen-bond with Asn290 in both ferric- complex with the substrate superoxo and ferryl-oxo intermediates (Fig. S5 and S6 in ESI†).
As a result of O2 activation, a ferryl-oxo [Fe(IV)-oxo] intermediate Whilst analysis of the long-range correlated motions in the (IM2 in Fig. 1), is generated in a manner established for other Fe(IV)]O complex (IM2) show a similar pattern as those for the 2OG oxygenases (see ESI† for details of calculations). The Fe(IV)- ferric-superoxo complex (Fig. S3†), there are differences. Over- oxo intermediate, IM2, abstracts a hydrogen from the substrate all, compared to the ferric-superoxo complex, the intensities of methyl group to give the radical cation IM3, which undergoes the correlated and anticorrelated motions in the Fe(IV)]O rebound to give the hydroxylated intermediate PD. To investi- complex (IM2) show lower intensities, e.g. in the N-terminal gate the role of protein exibility in reactivity of Fe(IV)-oxo region (residues 7–200, including the Fe-ligands His188 and intermediate IM2,a1ms simulation was performed (Fig. 5). The Glu190), and in the C-terminal region aer residue 308 ground state of Fe(IV)-oxo intermediate IM2 in the non-heme (Fig. S3†). iron oxygenases is in a high spin quintet state (S ¼ 2, M ¼ 5), as shown by studies on model complexes and other non-heme Creative Commons Attribution-NonCommercial 3.0 Unported Licence. 35 3.3 The rate of hydrogen atom transfer as a function of enzymes. The Fe(IV)-oxo intermediate prefers to abstract – s* protein exibility and geometric determinants a substrate methyl hydrogen through an (Fe O) z2 antibonding orbital due to exchange-enhanced reactivity, as shown by Shaik Hydroxylation begins with hydrogen atom transfer (HAT) fol- and coworkers.36 An electron shi diagram showing hydrogen lowed by a rebound process (IM2 to IM3 to PD in Fig. 1). To s* atom transfer (HAT) through z2 is given in Fig. 5a. The results evaluate the in uence of protein dynamics on the reactivity of 3 imply that linear positioning of a substrate N -methyl lysine Fe(IV)-oxo intermediate, we carried out reaction modeling using methyl group with respect to the Fe–O bond is important for ve snapshots from the MD trajectory. The Fe–O–N angle efficient catalysis. Thus, the Fe–O–N bond angle (Fig. 5c) is during dynamics ranges around 150 10 (Fig. 5). The potential
This article is licensed under a a geometric determinant for HAT. The proximity of the methyl energy prole for the reaction from a snapshot at 443 ns is given group hydrogens to the oxygen of Fe]O is another factor that in Fig. 6b. The barrier at the QM(B2)/MM level of theory with will affect the barrier for reaction. Another possible electron zero-point energy correction (QM(B2+ZPE)/MM) is Open Access Article. Published on 04 September 2020. Downloaded 10/2/2021 10:32:33 PM.
Fig. 5 Substrate dynamics for the Fe(IV)-oxo intermediate. (a) Fe(IV)-oxo intermediate with the substrate and MO diagram for a s-trajectory HAT. (b) Distance of the Fe(IV)-oxo O to substrate N during the 1 ms simulation. The y-axis is distance (A)˚ and x-axis is time (ns). (c) Variation in the Fe– O–N angle for the 1 ms simulations. The y-axis is the angle ( ).
9954 | Chem. Sci.,2020,11, 9950–9961 This journal is © The Royal Society of Chemistry 2020 View Article Online Edge Article Chemical Science