Lysine‐241 Has a Role in Coupling 2OG Turnover with Substrate Oxidation During KDM4‐Catalysed Histone Demethylation

Abstract The JmjC histone lysyl demethylases (KDMs) play important roles in modulating histone methylation states and have the potential to be regulated by oxygen availability. Lys241 of the KDM4 subfamily is proposed to be important in oxygen binding by KDM4A. We report evidence that, although Lys241 is unlikely to be directly involved in oxygen binding, it has an important role in coupling 2‐oxoglutarate cosubstrate oxidation with lysine demethylase activity. The results suggest that compounds promoting the uncoupling of substrate oxidation are of interest as JmjC‐KDM inhibitors.

H3K9, H3K36 (KDM4A-C) and H1.4K26 (KDM4A-D). [6] KDM4A/E have also been reported to catalyset he demethylation of Nmethylated arginines in the context of histonef ragment peptides. [7] Abnormal expression patterns of JmjC-KDMsh ave been linked to multiple diseases and disorders including in autism, [8] cardiach ypertrophy, [9] mid-line defects [10] and several cancers. [11] Consequently,m odulating KDM activity is of medicinal interest. However, therapeutic targeting of KDMs is currently compromised by an incomplete understanding of their biochemicala nd physiological roles. For example, it is possible that KDM catalysis is regulated by the availability of O 2 within some cells, althoughe vidence for the physiological relevance of such regulationi si ncomplete. [12] X-ray crystal structures of KDM4 enzymes in complex with substrate peptides have been reported and provided insights into mechanism and ab asis for inhibition studies. [1] An early crystallographic and mutagenesis study on KDM4A by Chen et al. identified ac atalytically essential active-site lysine residue (K241) that was proposed to regulate O 2 binding (Scheme 1); [13] hence K241 is potentially involved in regulating the O 2 sensitivity of KDM4s. K241, whichi sc onserved within the KDM4 subfamily but absent in other KDMs,i st herefore an attractive target residuefor KDM4-selective inhibitors.
Here, we report biochemical studies assessing the role of K241 in KDM4Ac atalysis. The resultsr evealt hat the K241A KDM4A variant is capable of catalysing substrate-uncoupled oxidative decarboxylation of 2OG at comparable efficiency to wild-typeK DM4A, thus implying as imilarly efficient reaction with O 2 (at least for uncoupled turnover). Demethylationo fa trimethylated H3K9 peptide by K241A KDM4A was also observed, although the catalysis was markedlyl ess efficient than for wild-type KDM4A,a nd demethylation activity wasn ot increased upon increasingt he O 2 concentration. Binding analyses by fluorescencep olarisation indicate similar binding efficiencies of the histone substrate to both the WT enzyme and the K241A variant. Overall, the results imply that K241 is important in KDM4A catalysis;a lthough it does have ar ole in coupling 2OG oxidation and demethylation, it is unlikelyt ob e involved in regulating O 2 binding.
Initially,w ep roduced two recombinant variants of KDM4A (residues 1-359) in Escherichia coli BL21(DE3) cells;atruncated wild-type sequence (KDM4AW T), or av ariant with an alanine in place of Lys241 (K241A KDM4A). Studies were then carried out to evaluatet he ability of the two KDM4A proteins to catalyse the demethylation of ah istonep eptides ubstrate. The two [ proteins (1 mm)w ere individually incubated with 2OG (100 mm, ac oncentration well above its WT K M value of 26 AE 7 mm), [12b] ascorbate (100 mm), ferrous iron (10 mm)a nd a1 5-residue H3K9me3 peptide( 100 mm,s equence:A RTKQTARKme3STGG-KA) in HEPES buffer (50 mm,p H7.5) at 37 8C; the extento ft he reactionw as determined by using MALDI-TOF mass spectrometry after 40 min. Substantial demethylation of the H3K9me3 peptidewas observed in the sample with WT KDM4A, resulting in formation of both di-and monomethylatedp roducts (H3K9me2 and H3K9me1 respectively,F igure 1A). The K241A variant,h owever,m anifested only minimal KDM activity;n o H3K9me1 or unmethylated peptide( H3K9me0) was evident, althoughl ow levels of H3K9me2w ere observed ( Figure 1A). Demethylation was not increased by increasing the concentration of 2OG to 2mm (Figures S1 and S2 in the Supporting Information). These findings suggest that K241 is required for efficient KDM activity,t hus supporting previous work that also employed MS. [13] Minimal K241A KDM4A-catalysedd emethylation activity was observed with other histonef ragmentp eptides reported to be KDM4A substrates ( Figure 1B).
Given the role of K241 in O 2 binding/recruitment proposed by Chen et al., [13] we postulated that the lack of KDM activity might be due to reduced binding of O 2 . 1 HNMR analyses were therefore carriedo ut to assess the ability of the WT and K241A KDM4A to catalyse substrate-uncoupled turnover of 2OG to succinate, ap rocess that requires O 2 binding. Samples containing either WT or K241A (20.5 mm), an excesso f2 OG (2 mm), ascorbate( 1mm), and ferrous iron (100 mm)i na mmonium formate buffer (50 mm,pH7.5) were subjected to 1 HNMR analysis at 1.5 min intervals over a3 0-min reaction. With both WT and K241A KDM4A, the triplet 1 Hr esonance at d H = 2.4 ppm (corresponding to 2OG) decreased in intensity during the analysis, while an ew singlet 1 Hr esonance at d H = 2.3 ppm, corresponding to succinate emerged ( Figures 1C and S3). The initial succinate formation rates were neari denticalw ith both enzymes (WT = 1.35 mm min À1 ,K 241A = 1.55 mm min À1 ,F igure 1C,n ote: 2OG turnover was too slow for accurate determination of the Michaelis-Mentenk inetic parameters). Uncoupled 2OG turnover by the WT and K241A enzymes was inhibited by the 2OG competitor N-oxalylglycine;i nterestingly,i nhibition was more  [14] which is proposed to interactw ith K241 in the KDM4Aa ctive site (inferred from modelling studies). [15] Results from NMR bindinge xperiments support preferential binding of JIB-04 to the K241A variant over the WT enzyme( provisional K D(JIB-04) = 22 AE 5a nd 7 AE 2 mm for WT and K241A, respectively,F igures 1D andS 6), thusi mplying no/suboptimal interactions between JIB-04 and K241. Experimentsw eret hen conducted in the presence of ah istoneH 3e ight-residue fragment peptidec ontaining N e -trimethyllysine at position9 (H3K9me3, sequence:A RKme3STGGK). Initial succinate formation rates were markedly increased for both the WT and K241A enzymes; this implied binding of the histone fragment, whichisr eported to stimulates uccinate formation by KDM4, [16] and potentially demethylation. The 1 Hr esonances corresponding to lysyl methyl groups were obscured by residual glycerol and HEPES from the enzyme stocks;t his hindered the detection of KDM activity.S timulation of succinate formation wasg reater for WT than for K241A KDM4A.
MS studies were then carried out to investigate whether demethylation by K241A KDM4Ai ss timulated by increasing the O 2 concentration. KDM assays (using the same final concentrations of components as above)w ere carried out with the use of aM ass Flow Controller (Brooks Instruments) to equilibrate the reactions at either 20 or 80 %O 2 ,asd escribed; [12b] reactions were left for 10 min before quenching (with methanol) and MALDI-TOF MS analysis. As reported,t he KDM activity of WT KDM4A increased with increasing O 2 concentration (from 31 to 46 %d emethylation, Figure 1E). [12b] However, only trace demethylation (< 5%)w as observed in the samples with K241A KDM4A at either 20 or 80 %o xygen ( Figure 1E); this indicated that oxygen binding/recruitment does not limiting the KDM activity of the K241A variant.
The crystal structures of KDM4A appear to show that K241 interactsw ith the H3 fragment peptides ubstrate (Scheme 1,  [17] we therefore investigated whether the KDM activity of the K241A variant is limited by weakened substrate binding. The binding of WT andK 241A KDM4At oahistone fragment peptidew as evaluated in af luorescencep olarisation assay.A 15-residue H3K9me3 peptideC -terminally labelled with af luorescein fluorophore, that is, ARTKQTARKme3STGGKA-fluorescein, was found by MALDI-TOFM St ob eaW TK DM4A demethylation substrate (a mass shift of À14 Da was observed under standard conditions). Thel abelleds ubstrate (20 nm)w as incubated with the 2OG analogue N-oxalylglycine (1 mm), [18] NiCl 2 (10 mm)a nd variousc oncentrations of the WT and K241A enzymes( 0-20 mm). The fluorescencee mission spectra of the samples parallel and perpendicular to the excitation plane were then measured, and used to calculate the fluorescence polarisation at each enzyme concentration. The apparent binding constants for peptideb inding were near identical for both enzymes (3.06 AE 2.9 mm for WT KDM4A, and 4.54 AE 2.1 mm for K241A KDM4A, respectively), thus indicating that mutation of the K241 residue to alanine does not alter the affinity of KDM4A for the peptides ubstrate ( Figure 1F).
The overall MS, NMR spectroscopy and fluorescencep olarisation studies imply that K241 has ar ole in KDM4A catalysis. However, the observation that the K241A variant catalyses uncoupled succinate formation with comparablee fficiency to the WT implies that the role(s)o fK 241d uring catalysis do not involve( at least initial) bindingo fO 2 . [13] Thus, the observed mixed-modei nhibition [14] of K241A by JIB-04 probablyd oes not reflect direct disruption of O 2 binding.S uccinate formation by the K241A variant is stimulated by ah istone fragment peptide, and af luorescently labelledh istone peptide binds in a similarly tight mannert ot he WT and K241A KDM4A. Thus, K241 appears to have ar ole in coupling 2OG turnover to substrate oxidation. One possibility is that K241 helps to orientate the methylated lysyl side chain within the KDM4A active site, thereby ensuring efficient oxidation of am ethyl CÀHb ond after succinate formation.K 241m ight also limit the access to the active site of water molecules that could quencht he reactive iron(IV)-oxo intermediate before methylC ÀHb ond oxidation can occur.T argeting K241 is of interestb oth with respect to developing classical tightly binding inhibitors and to developing inhibitors that decouple 2OG and substrate oxidation. Such inhibitors are of interest because uncoupling could cause inactivation;i nt he absence of "prime" substrate, self-oxidation of 2OG oxygenases can occur,e ither directly or throught he production of reactive oxygen species. [19] Therefore, molecules that bind and induce self-oxidation before dissociating, for example, K241 binders, have the potential to be catalytic inactivators.