Structural Basis of Histone Demethylase KDM6B Histone 3 Lysine 27

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The structural basis of the histone demethylase KDM6B histone 3 lysine 27 specificity Sarah Elizabeth Jones, Lars Olsen, and Michael Gajhede Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01152 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 11, 2017

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Biochemistry

The structural basis of the histone demethylase KDM6B histone 3 lysine 27 specificity

Sarah E. Jones1, Lars Olsen1 and Michael Gajhede1*

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Biostructural Research, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark

Keywords: Histone demethylase, KDM6B, x-ray crystallography, substrate binding

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ABSTRACT

KDM subfamily 6 enzymes KDM6A and KDM6B specifically catalyse demethylation of di-/trimethylated lysine on Histone 3 lysine 27 (H3K27me3/2) and play an important role in repression of developmental genes. Despite identical amino acid sequence in the immediate surroundings of H3K9me3/2 (ARKS) the enzymes do not catalyse demethylation of this general marker of repression.

In order to address this question for KDM6B we used computational methods to identify H3(1733) derived peptides with improved binding affinity, that would enable co-crystallization with the catalytic core of human KDM6B (ccKDM6B). A total of five peptides were identified and their IC50 values were determined in a MALDI-TOF based assay. Despite none of the peptides showing showed significantly higher affinity compared to the H3(17-33) peptide, it was possible to co-crystallize ccKDM6B with a H3(17-33)A21M peptide. This structure reveals the interactions between the KDM6B zinc binding domain and the H3(17-23) region. Although KDM6A and KDM6B differ in primary sequence, particularly in the H3L20 binding pocket of the zinc binding domains, where to histidines in KDM6A have been replaced by a glutamate and a tyrosine, they bind the H3(17-23) in a very similar fashion. This structure shows that KDM6B, in analogy with KDM6A, also uses the zinc binding domain to achieve H3K27me3/me2 specificity. The histidine to glutamine substitution at amino acid position 1564 in the KDM6B zinc binding domain can further rationalize, why KDM6B binds substrates with higher affinity than KDM6A.


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Biochemistry

INTRODUCTION The accessibility of genes to the cells transcriptional machinery is known to play an important role in gene regulation [1]. This accessibility is to a wide extent controlled by posttranslational modifications (PTMs) of the unstructured N-terminals of the histones, where the most important modifications are acetylations and methylations of lysine residues [2]. The large number of enzymes that govern the genome PTM patterns are classified as readers, writers or erasers dependent on whether they read, introduce or remove PTMs. Some erasers form the lysine demethylase (KDM) family, which comprises a total of 24 members [3]. The enzymes can be subdivided into subfamilies that have specific substrates on the histone tails [4]. With exception of KDM1A and KDM1B, all of the enzymes belong to the family of Jumonji C (JmjC) domain containing, iron and 2-oxoglutarate (2-OG) dependent oxygenases [5]. Generally the KDM family is known to be heavily involved in developmental control and cell fate decisions [4]. Consequently, they are also implicated in the development of many forms of cancer [6]. The KDM6 subfamily members KDM6A (UTX) and KDM6B (JMJD3) catalyse demethylation of tri- and di-methylated lysine 27 on Histone 3 (H3K27me3/2) and have been shown to be involved in developmental HOX gene regulation [7]. KDM6B has also been shown to control inflammatory responses [8]. The subfamily comprises a third member KDM6C (UTY) that, however, has been shown to have a reduced H3K27me3/2 demethylase activity [9]. Initially the specificity of the KDM6 family has been puzzling. The H3 sequence motif surrounding both K9 and K27 are A-R-Kme-S so it was not known why the subfamily only catalyses the demethylation of K27.


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A number of studies have addressed the structural basis of the specificity of the KDM6 subfamily demethylation reaction. The crystal structure of a truncation of human KDM6A (ccKDM6A) in complex with a H3 residues 17-33 (H3(17-33)) derived peptide has been reported. It was found that the JmjC domain interacts with H3 residues 25-33 and, in addition, a subfamily conserved zinc-binding domain bound residues 17-21 [10]. In particular, binding of residues H3R17, H3L20, H3R26, H3A29, H3P30 and H3T32 were by mutational analysis found to be important for the demethylation reaction. It was also pointed out, that the zinc-binding domain changed conformation upon binding of the H3 peptide, a change that allowed for recognition of H3L20 by an otherwise buried hydrophobic patch on KDM6A. These findings explained the ability of KDM6A to selectively demethylate H3K27me3/2. The crystal structure the catalytic core of mus musculus KDM6B in complex with a H3(24-34) peptide has also been determined [8]. Due to the shorter N-terminal of the complexed peptide it has not been possible to determine whether KDM6B uses the same mechanism as KDM6A to achieve K27 selectivity. Higher KM values for Histone 3 peptide substrates of KDM6A compared to KDM6B have previously been reported [8,11]. Despite this, we or others have not been able to crystallize a KDM6B:H3(17-33) complex. Consequently, we have used a computational method approach to identify H3(17-33) derived peptides with even better binding affinity. Although none of the identified variant peptides showed significantly better binding affinity than the H3(17-33) peptide one of them crystallized with ccKDM6B. Here we report the crystal structure of a truncation of human C-terminal JmjC and zinc-binding domains of human KDM6B 1141-1683 (del 1637-1675)-6H (ccKDM6B) with bound 2-OG and Fe in complex with a derived peptide H3(17-33)A21M. From this structure we show that KDM6B uses a similar mechanism of substrate recognition to that of KDM6A to achieve H3K27me3/2 specificity.


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Biochemistry

MATERIALS AND METHODS KDM6B model A homology model of KDM6B 1141-1863 (with deletion 1637-1675)-6H was created in Maestro 9.8 homology modeling module Prime [12] using PDB ID 4EZH as a template. The histone 3 peptide H3(17-33) was added to the model by aligning the KDM6B homology model with the crystal structure of the catalytic domain of KDM6A with bound H3(17-33)K27me3 (PDB ID 3AVR), and extracting the peptide into the KDM6B model. This model was modified by changing Ni(II) to Fe(II), and changing the histone peptide K27me3 to a non-methylated lysine. To refine the KDM6B-peptide interactions, a 12ns Molecular dynamics (MD) simulation was performed according to the default protocol, followed by a minimization of the last frame, both using the Desmond package [13]. This model was used for further calculations. See detailed methods in supplementary material.

FoldX PositionScan The program FoldX [14] was used to identify potential favorable sequence variants of the histone peptide by generating differences in binding energies between the wild-type residues and all 20 natural amino acids. Both the structure of mouse KDM6B-H3(24-34) complex (PDB ID 4EZH) and complexed KDM6B-H3(17-33) model were first optimized by using the RepairPDB function of FoldX. Next, the PositionScan function of FoldX was used to scan each residue in the H3 peptide. This provides an output of free energy of binding (kcal/mole) for the wild-type residue and for each sequence variant. The difference in binding energy between the wild-type and variants were calculated and the 5 most favorable sequence variants in both crystal structure and model were chosen for further investigation.


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Molecular dynamics (MD) and Free Energy Pertubation (FEP) calculations The FoldX results were analyzed further with MD and FEP. Therefore analysis of 12ns MD simulations and FEP calculations were done to validate these results. Detailed methods can be found in supplementary. MD simulations, FEB calculation and Time Event Analysis were performed using the Desmond package (version 3.8) [13] or VMD (version 1.9.1) [15].

Expression and purification of KDM6B 1141-1683 (del 1637-1675)-6H Human KDM6B 1141-1683 (del 1637-1675)-6H (NCBI ref.: NM_001080424, UniProt Accession ID: O15054-1) was cloned into a pFB1 vector (Genscript) and expressed in Spodoptera fugiperda (Sf9) cells. Sf9 cells were cultivated in sf9 media supplemented with fetal bovine serum and penicillin Streptomycin at 27oC, 130 rpm. The culture was infected with P3 recombinant Baculovirus at a multiplicity of infection of 3 and incubated at 27oC, 130rpm for 72 hrs. Cells were harvest at 7800 xg, and re-suspended in lysis buffer (20mM Tris pH 8.5, 300mM NaCl, 10% Glycerol, 10mM (NH4)2Fe(SO4)2, Protease inhibitor tablets (Roche) (1 tablet/50mL buffer)). Cells were homogenized at 2 kbar and centrifuged at 87.200 xg for 2 hrs. Supernatant was filtered on a 0.45µm filter and loaded on a 5mL HisTrap FF column. The column was washed with 10CV HisTrap binding buffer (20mM Tris pH 8.5, 300mM NaCl, 10% Glycerol, 10mM (NH4)2Fe(SO4)2) followed by 8% HisTrap elution buffer (20mM Tris pH 8.5, 300mM NaCl, 10% Glycerol, 10mM (NH4)2Fe(SO4)2, 250mM Imidazole). Protein was eluted from the column using a linear gradient of 8-100% 250mM Imidazole over 20 CV. Protein eluted between 24% and 60% Imidazole. Eluted KDM6B protein was diluted 20-fold in IE binding buffer (20mM Tris pH 8.5, 10% Glycerol, 10mM (NH4)2Fe(SO4)2) and loaded onto a 5mL HiTrap Q


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Biochemistry

HP column. Column was washed with 10CV IE binding buffer. Protein was eluted using a linear gradient of 0-100% 1M NaCl over 20CV. Protein eluted between 35% and 45% 1M NaCl. The eluted protein was concentrated to approx. 3mL and loaded onto a Superdex 200 26/60 column equilibrated with SEC buffer (20mM Tris pH 8.5, 150mM NaCl, 5% Glycerol, 0.5mM TCEP, 2mM 2-OG 10mM (NH4)2Fe(SO4)2). Protein eluted at 195mL, was concentrated, flash frozen in liquid nitrogen and stored at -80oC until further use. Protein concentration was measured with UV-vis (ε280nm = 98.320 M-1 cm-1)

Crystallization of KDM6B in complex H3(17-33)A21M peptide Initial crystals were grown in a 200nL drop with the protein sample 6mg/mL ccKDM6B, 2mM H3(17-33)A21M in 20mM Tris pH 8.5, 150mM NaCl, 5% Glycerol, 0.5mM TCEP, 2mM 2-OG, 10µM (NH4)2Fe(SO4)2 and the precipitant 0.1M Tris pH 8.5, 25%(v/v) tert-butanol in ratio 1:1 and 2:1, sitting drop at 4oC. This was done at the High Throughput Crystallization Laboratory (HTX Lab) at the EMBL in Grenoble, France [16]. Crystallization conditions were optimized and crystals for data collection was grown in 0.1M Tris pH 7.5, 15%(v/v) tert-butanol, 1:1 sample-precipitant ratio (2uL drops), sitting drop at 4oC. Crystals appeared after 3 days. The crystal was then serial transferred into mother liquor supplemented with 15%v/v EthyleneGlycol and flash frozen and stored in liquid nitrogen.

Data collection and structure determination Data sets were collected at the ID23-2 (GEMINI) beamline [17] and the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. The data was processed automatically using XDS [18] and further scaled and merged using Aimless [19]. The structure of ccKDM6B with


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bound H3(17-33)A21M was solved by molecular replacement in Phaser [20] using the structure of ccKDM6A with bound H3(17-33)K27me3 peptide (PDB ID: 3AVR) as a search model. Structure was refined using program Phenix1.10.1-2155 [21]. The structure has been deposited in the PDB as ID 5OY3.

Dose-Response Assay ccKDM6B demethylation reactions for MALDI-TOF analysis consisted of 2.5µM enzyme, 20µM tri-methylated Histone 3 peptide H3(14-34)K27me3, 2-5000µM inhibitor peptide (H3variant), 50µM 2-OG, 100µM ascorbate, 100µM (NH4)2Fe(SO4)2, in 50mM HEPES pH 7.5, 150mM NaCl buffer. Components were premixed in two batches. The first batch containing enzyme, inhibitor, Fe(II), and ascorbate. The second batch containing 20µM H3(14-34)K27me3 and 50µM 0.5mM 2OG. Batch 1 was incubated on ice for 15min, before initiating the reaction by mixing with batch two. Reactions were incubated at 37oC for 8min and then quenched 1:1 with 3% formic acid. 1µL reaction mixture was spotted onto a MALDI target plate (Bruker) overlaid with 1µL matrix (α-cyano-4-hydrocinnamic acid in MeOH-H2O-TFA 550:475:25), and data was collected on a microflexTM mass spectrometer (Bruker Daltronik, inc.), and spectrum were analyzed with flexAnalysis Version 3.4 (Bruker Daltronik, inc.).


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Biochemistry

RESULTS AND DISCUSSION To find a sequence variant of the histone peptide that would enhance the binding affinity to ccKDM6B, a FoldX PositionScan, which gives a fast estimation of binding energy of all natural amino acids at a given position, was performed. This was done for all positions of the peptide in both the crystal structure H3(24-34) peptide of the mus musculus ccKDM6B:H3(24-34) complex (PDB code 4EZH) and a homology model of the H3(17-33) peptide of human ccKDM6B:H3(1733) complex (Full FoldX results in figure S1 and S2 in supplementary). From the FoldX results, the five sequence variants of the histone peptide with the highest gain in binding energy for each system were chosen for further analysis. For the mouse ccKDM6B:H3(24-34) complex crystal structure, the variants were A25L, A25M, A25K, A29L, A29L had the largest gain in binding energy, whereas for the model of human ccKDM6B:H3(17-33) complex the peptide variants A21M, A24R, A24M, A24H and T32M had the largest gain in binding energy. Further analysis of 12ns MD simulations and FEP were performed to further validate these results of which H3(17-33)A24R showed not likely to be favorable (results in supplementary figure S3 and S4). Dose-Response curves were generated with a MALDI-TOF activity assay for H3(24-34) and H3(17-33) derived peptides. The H3(24-34) peptides had no inhibitory effect on activity in the concentration range 0-5000µM. The IC50 values for H3(17-33) was determined to 155µM. The variants H3(17-33)A24H and H3(17-33)T32M had higher IC50 values of 370µM and 650µM respectively. Peptide H3(17-33)A21M, H3(17-33)A24M and H3(17-33)A24R, showed slightly better IC50 values of 70µM, 50µM and 75µM respectively, however none of the mutated peptides resulted in significant gain in inhibition of the protein relative to the H3(17-33) peptide. Dose-response curves are seen in figure 1. Despite this, crystallization trials were undertaken for


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ccKDM6B with the best binding H3(17-33) derived peptides. This led to crystallization and structure determination of a ccKDM6B:H3(17-33)A21M complex.

Figure 1. ccKDM6B dose-response curves for H3(17-33) peptides. Data points were produced using a MALDI-TOF activity assay.

Overall structure The crystal structure of ccKDM6B in complex with 2-OG, Fe and H3(17-33)A21M was solved at 2.1Å resolution (data collection and refinement data can be seen in table 1). Electron density is visible for residues 1157-1639 and the electron density is seen for all amino acids in the histone peptide H3(17-33), with exception of the side chains of R17, K18 and residue G33 which are less ordered.

Table 1: Summary of data collection and refinement statistics Values for the outer shell are given in parenthesis Diffraction Source

ESRF beamline ID23-2


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Biochemistry

Wavelength (Å)

0.873

Temperature (K)

80

Detector

Marmosaic 22mm CCD

Crystal-detector distance (mm)

215.98

Rotation range per image (o)

0.05

Total rotation range (o)

180

Space Group

P41212

a, b, c (Å); α,β, γ (degrees)

68.9, 68.9, 228.5; 90, 90, 90

Mosaicity (o)

0.14

Resolution range (Å)

48.72-2.14

Total no. of reflections

343768(33112)

No. of unique reflections

31653(2951)

Completeness (%)

99.7(97.1)

Redundancy

10.9(11.2)

(I/σ(I))

12.2(1.88)

Rr.i.m.

0.182(1.457)

Over B factor from Wilson plot (Å2)

31.4

Refinement Resolution range (Å)

48.73-2.14

Completeness (%)

99.9

σ cutoff

F > 1.34σ(F)

No. of reflection, working set

31572(2012)

No. of reflections, test set

2000(136)

Rwork/Rfree

0.1650/0.2207

Cruickshank DPI

0.186 Å

No. of non H-atoms


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Protein/peptide Ligand/Ions/water

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3622/125 10/2/300

Average B factors Protein/peptide

41.1/45.0 37.0(AKG)/24.8(Fe), 33.2(Zn)/39.8

Ligand/Ions/water Ramachandran plot Favored Allowed Not allowed

96.7% 3.3% 0.0%

RMSD bond length (Å)/Angle(°)

0.012/1.11

The structure consists of three conserved domains, the JmjC domain (residues 1157-1485) with a beta sheet jelly roll fold with small scaffold alpha helixes, the linker domain (residues 14901558 and 1623-1635) comprising of four alpha helices and the zinc binding domain (residues 1563-1620). This fold is the same as seen previously for all structures of the KDM6 family. Comparing the structure to that of human ccKDM6A in complex with H3(17-33)K27me3 (PDB code 3AVR [10]) the alignment of backbone Cα gives a RMSD of 1.01Å. The main difference between the two complexes is the short alpha helix residues 1602-1610 in KDM6B and 1358-1366 in KDM6A located within the zinc binding domain, where the C-terminal end of the helix in KDM6A is angled outward by about 10 degree compare to that of the helix in KDM6B. Therefore, there is also a slight shift in the Zinc atom, and residues 22, 23 and 24 in the H3 peptide that are in closest proximity to the zinc atom. However, crystal packing of KDM6B may also contribute to this distortion, as the neighboring molecule within the crystal lay adjacent to the zinc binding domain.


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Biochemistry

It has been seen for KDM6A that there is a conformational change of the zinc binding domain upon binding of the H3(17-33) peptide [10]. However, comparing this structure of ccKDM6B to previously solved structure of non-peptide bound human ccKDM6B (PDB code 2XUE [8]), the overall conformation does not change upon binding of the histone peptide (Figure 2b), and alignment of backbone Cα gives a RMSD of 0.57Å. It is possible that this could be an effect due to the ccKDM6B deletion 1637-1675 which is rich in arginine and proline. However this deletion causes KDM6B to be a similar length and gives higher sequence similarity to KDM6A and would be located on the opposite side of the zinc binding domain compared to the peptide binding cleft. Also the structure of mouse ccKDM6B in complex with a shorter histone peptide H3(24-34) (PDB code 4EZH [8]) has the same overall fold and alignment of backbone Cα gives a RMSD of 0.43Å. See figure 2 for alignments.


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Figure 2. Alignment of backbone carbon in catalytic domains of ccKDM6B:H3(17-33)A21M complex (green) and (a) human catalytic core KDM6A:H3(17-33) complex (pink, PDB code 3AVR[10]) RMS 0.69Å, (b) human catalytic core KDM6B (yellow, PDB code 2XUE [8]) RMS


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Biochemistry

0.57Å and (c) mouse catalytic core KDM6B:H3(24-34) complex (blue, PDB code 4EZH [8]) RMS 0.42Å.

Binding of H3(17-33)A21M peptide The backbone of all 21 amino acids in the H3(17-33) are visible in the structure of the complex. The peptide residues 17-25 bind and span across the zinc-binding domain and the remaining residues 25-33 bind to the catalytic Jumonji domain with lysine 27 oriented toward the catalytic cavity where the iron and 2-OG are situated. The binding of this peptide is very similar to that seen in the structure of the mus musculus ccKDM6B with H3(24-34) and in the structure of human ccKDM6A in complex with H3(17-33). For both ccKDM6B and ccKDM6A complex structures the electron density for the peptide Nterminal amino acid R17 is poor, but in both cases the side chain points toward a mainly hydrophilic pocket lined with Q1564, G1565, R1566, V1567 and E1570 in KDM6B (figure S5a) and H1320, G1321, R1322, T1323 and E1226 in KDM6A. It has been found by mutational studies that recognition of R17 through E1226 in KDM6A is important for demethylation [10]. This residue is conserved as E1570 in KDM6B. The electron density for the side chain of K18 is also poor in both structures. This may be due to a mainly hydrophobic pocket in both enzymes - Q1564, V1588, Y1598, V1617 and L1619 in KDM6B (see figure S5b) and H1320, V1344, Y1354, V1373 and L1375 in KDM6A and therefore there are no obvious interaction. The backbone is held in place by hydrogen bonding to the side chain of KDM6B residue Q1564, through both the NH2 amide and CO carbonyl groups. This corresponds residue H1320 in KDM6A that hydrogen bonds to the backbone carbonyl only.


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This additional interaction could possibly explain the approximately seven fold lower KM of KDM6B compared to KDM6A. In both KDM6A and KDM6B structures Q19 binds through hydrogen bonds from the backbone carbonyl to the protein backbone amide of Y1598 in KDM6B and Y1354 in KDM6A, and from the peptide backbone amide to the backbone carbonyl of N1596 in KDM6B and K1352 in KDM6A.

Furthermore, the Q19 side chain amide moiety forms hydrogen bonds to a

backbone amide and carbonyl of N1264 on the neighboring protein molecule in the ccKDM6BH3(17-33) complex crystal, suggesting that this residue contributes to crystal packing (see figure S5c). L20 has previously been found to be important for substrate recognition in KDM6A [10], The L20 side chain sits within a hydrophobic pocket constituted of Q1564, Y1573, L1586, V1588, Y1598, V1600, L1619 in KDM6B and H1320, H1329, L1342, V1344, Y1354, V1356, L1375 and in KDM6A, where the major differences are the replacements of two histidines in KDM6A with glutamine and tyrosine. It was found for ccKDM6A that binding of the H3(17-33)K27me3 peptide caused a conformational change in the zinc-binding domain, where the Y1354 residue is displaced, exposing this hydrophobic pocket [10]. Interestingly, it has before been observed that the conformation of ccKDM6A in complex with a H3(17-33) peptide is similar to that of ccKDM6B with and without bound H3(24-34) peptide [8]. It is also observed here that there is no conformational change upon binding of the histone peptide in ccKDM6B, and therefore the hydrophobic pocket is exposed in both non-peptide bound and peptide bound structures. The L20 hydrophobic binding pocket can be seen in figure 3.


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Biochemistry

Figure 3: The L20 hydrophobic binding pocket (a) ccKDM6B (green) and (b) to catalytic core KDM6A (pink).

In position 21 of the histone peptide the alanine has been mutated to a Methionine in the ccKDM6B:H3(17-33) complex structure, see figure 4. However in both ccKDM6B and ccKDM6A structures both M21 and A21 residues form non-polar interactions with side chains of


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T1597 and L1599 in KDM6B and T1353 and I1355 in KDM6A. In both structures the peptide backbone carbonyl hydrogen binds to the backbone amide of Y1598 and Y1354 in KDM6B and KDM6A respectively. The M21 side chain of the Histone derived peptide is located within 5Å of protein residues I1440, D1442 and D1443 on the neighboring molecule within the crystal. Furthermore, the side chain hydroxyl of KDM6B T1596 hydrogen bonds to side chain carboxyl of N1442 on the neighboring molecule. Notably, this interaction seems to be very important for crystal packing as an alanine at this postionhinders crystal growth under otherwise identical conditions.

Figure 4: Binding of H3(17-33)A21M peptide residue M21 to ccKDM6B (green). Neighbouring molecule in crystal at interface by H3M21 (wheat).

The side chain of residue T22 is located within 4Å of KDM6B residues P1571, A1572, Y1573 (see figure S5d). In comparison T22 resides up to 5.5Å away from corresponding KDM6A residues P1327, A1328, H1329.


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Biochemistry

In the ccKDM6B structure the electron density clearly shows that the histone K23 residue is directed towards a hydrophilic pocket comprised of residues N1576, D1579 and K1509, see figure 5.a. Similar binding is possible to corresponding KDM6A residues K1265, E1335 and S1332, however in this structure the electron density for K23 is very poor, indicating that the side chain of residue is not fixed in this structure, see figure 5.b.


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Figure 5: Binding of H3(17-33) peptide residue K23 to (a) ccKDM6B, clear electron density is seen for the side chain of K23 indicating a fixed position, and (b) to catalytic core KDM6A, election density is not clear for the side chain of K23, indicating flexibility of this side chain.

Moving from the zinc binding domain towards the Jumonji Domain, A24 of the Histone peptide does not have any obvious interactions with neither KDM6B nor KDM6A (figure S5e). The side chain of alanine 25 is directed towards a pocket with KDM6B side chain residues H1368, I1370, K1509, I1511 and Y1574, and the back bone carbonyl of the peptide A25 hydrogen bonds to the backbone amide of L1371 (see figure S5f). These interactions are identical in the mouse KDM6B:H3(24-34) structure, and the equivalent pocket is also seen in KDM6A with residues H1124, I1126, K1265, I1267, Y1330 with hydrogen bonding to I1127. R26 has also previously been found to be important for substrate specificity and peptide directionality for both KDM6B and KDM6A [8,10]. The aliphatic side chain is located by the side chain of L1371 in KDM6B and L1127 in KDM6A in a hydrophobic interaction. The clear electron density shows hydrogen bonding from the side chain guandinium moiety to KDM6B. The carboxyl moiety of D1333 binds to the Nε of R26. The carboxyl of E1244 binds to both Nη of R26 and the back bone carbonyl of N1331 binds to a single Nη. Equivalent bonds are seen to KDM6A residues D1089, E999 and N1087. However, these interactions are slightly different in the mouse ccKDM6B:H3(24-34) complex. As the electron density is not as convincing, probably due to the shorter peptide length, R26 has been modeled so the guandinium group is slightly shifted, where D1333 only hydrogen bonds to a single Nη, the side chain carboxyl of E1244 only binds to a single Nη and the back bone carbonyl of N1331 binds to both Nη (see figures S5g). Due to the more convincing electron density of this new structure, this suggests that binding of a


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Biochemistry

longer peptide that interacts with the zinc binding domain, enhances the interaction at this position. The side chain amine of K27 resides within the catalytic pocket lined with residues N1331, G1372, M1373, Y1379, and E1392 close to Fe (Nε->Fe 5.9Å) and 2OG (figure S5h). The peptide backbone carbonyl hydrogen bonds to the back bone amide of Q1377 and the backbone amide of K27 hydrogen bonds to the backbone carbonyl of L1371 and a water molecule. The same interaction are seen in the mouse ccKDM6B:H3(24-34) complex, however the distance between Nε and the catalytic iron is slightly less at 5.6Å. This is most likely because the lysine is trimethylated in this structure. Equivalent interactions are seen for KDM6A, and the Nε is even closer to the catalytic iron at 5.3Å. This may be due to the combination of the longer length of the peptide substrate and a trimethylated amine. The side chain of S28 in both ccKDM6B complexed structures point toward a hydrophilic pocket of E1244, R1246, R1272 and N1331 (figure S5i). Same enzyme residues are present in the ccKDM6A:H3(17-33) structure, however electron density clearly shows that the hydroxyl group of S28 points away from this pocket. Residue A29 is bound by hydrogen bonds to the side chains of R1246 and N1331 in both KDM6B structures (figure S5j). The same is seen for KDM6A. The backbone carbonyl of P30 hydrogen bonds to the Nε of R1246, and the side chain points toward P1388 in a pocket lined with P1388, T1434, G1435 and S1436 (figure S5k). These interactions are also found in KDM6A. P30 has before been found to be important for maintaining the conformation of the histone peptide and the Pro-Pro interaction has been found to be important for binding and directionality KDM6B and KDM6A. The side chain of A31


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points toward to Arginine residue, R1246 and R1272 in KDM6B (figure S5l) and R1001 and R1027 in KDM6A. The N-terminal end of the peptide in both structures of KDM6B and KDM6A in complex with H3(17-33) peptides have similar orientations. The main chain amide of T32 interacts with the main chain carbonyl of S1270 in KDM6B, and the side chain hydroxyl interacts with the main chain amide of S1270 (figure S5m). S1270 corresponds to S1025 in KDM6A and the same interactions are seen. In addition the side chain hydroxyl of T32 also hydrogen bonds to the amide of N1026 in KDM6A. KDM6B has a serine in this position. The N-terminal end of the H3(24-34) peptide, however, has a slight different orientation, so that the amide of T32 does not interact with KDM6B and only the side chain hydroxyl interacts with the main chain carbonyl of S1270. This could be due to the additional residue G34 or the shorter peptide length. The main chain carbonyl of G33 hydrogen bonds to the main chain amide in R1272 (R1025 in KDM6A) in all three structures (figure S5n), however the G34 does not have any interactions with KDM6B in the KDM6B-H3(24-34) structure. Here we have shown that the histone H3(17-33) derived peptide binds in a very similar way to KDM6B as to KDM6A. Residues R17, L20, R26, A29, P30 and T32 of the histone peptide were previously found to be predominantly important for KDM6A substrate recognition and directionality [10]. Interaction between these histone peptide residues and KDM6B are identical or highly similar. This strongly suggests that KDM6B uses the same mechanism as KDM6A to achieve H3K27me3/2 specificity. Specifically, in analogy with KDM6A, KDM6B also utilizes binding of the unique H3(17-23) binding motif RKQLATK to the zinc binding domain to distinguish the identical H3(7-10)K9me and H3(25-28)K27me ARKS binding motifs. Some subtle differences are observed that could explain the higher affinity of the histone substrate to


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Biochemistry

KDM6B. H1320 in KDM6A is replaced with residue Q1564 in KDM6B which increases hydrogen bonding to the K18-L20 region of the peptide. There also appears to be better binding of peptide K23 to KDM6B than KDM6A. The side chain of S28 may also hydrogen bond to KDM6B, whereas this interaction does not seem apparent in KDM6A.

Effects of Histone variant A21M on crystal packing The A21M variant of the histone peptide seems to facilitate crystal packing, as this sits in the interface between to enzyme molecules. Also crystallization in complex with other H3(17-33) derived peptides under the same crystallization conditions were attempted without result. To analyze this further, a 12ns MD simulation was done for both the crystal structure with the H3(17-33)A21M and for the crystal structure with the 21 position of H3(17-33) mutated to an alanine as in the wild type peptide. An analysis was done by counting the number of C-C and CS interactions, between the histone 21 side chain and KDM6B residues within 5Å, in every 1 in 10 frames of the 12ns MD simulations. This is shown both as the percent of frames which have interactions and the average number of interactions across all frames. Results can be seen in table 2. The presence of the methionine compared to alanine in the 21 position shows a vast increase in the degree of interaction to KDM6B residues T1597, Y1598 and L1599. This increase in interactions between H3M21 and KDM6B residues T1597, Y1598 and L1599 most likely stabilizes the orientation of T1597, enabling it to form a hydrogen bond to D1442 on the neighbouring molecule, which in turn would facilitate crystal packing. A time-distance analysis of the MD simulations also reveals a stable distance of approx. 2.7Å between the side chain hydroxyl of T1597 and the closest side chain carboxyl oxygen of D1442 (see time-distance analysis in figures S6 and S7). This analysis shows the relevance of a methionine in the 21


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position of the histone peptide for crystal packing and reveals a possible method for future crystallization of KDM6B with other H3(17-33) derived peptides. Table 2: Analysis of number of C-C and C-S interactions between H3A21/M21 and KDM6B residues within 5Å in every 1 in 10 frames in 12ns MD simulations. A21

M21

JMJD3 residues

% frames with contacts

Average no. of contact per frame

% frames with contacts

Average no. of contact per frame

N1576

2.0

0.02

3.1

0.03

G1592

10.3

0.1

10.7

0.1

T1597

1.6

0.02

42.5

0.8

Y1598

0.0

0.0

17.5

0.2

L1599

10.7

0.1

58.7

1.0

V1600

1.2

0.01

0.0

0.0

In conclusion, we have shown that a A21M variant of histone peptide H3(17-33) can facilitate the crystallization of a ccKDM6B-H3(17-33) complex. The structure of this complex reveals that KDM6B, in analogy to KDM6A, binds histone region H3(17-23) to the zinc binding domain to achieve substrate specificity. The structure further aids rational design of new KDM6 family selective inhibitors. It also reveals minor differences in the L20 and K23 binding pockets of KDM6A and KDM6B. These differences may be utilized in the design of inhibitors that are selective among the KDM6 enzymes. ASSOCIATED CONTENT


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Biochemistry

The following files are available free of charge. Supporting Information pdf-file, including Supplementory methods for MD simulations and Free Energy Perturbations, FoldX PositionScan Heat Maps, FEP data, Figures of histone peptide binding, Analysis of Distance between Thr1597 and Asp1442 at interface between molecules in PDB code 5OY3

AUTHOR INFORMATION Corresponding Author * Michael Gajhede, email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This project was supported by a grant from the Danish Research Council for Independent Research

and

by the

University of

Copenhagen

excellence

programme

CoNeXT

(www.conext.ku.dk). A large facility traveling grant from Danish Research Council for


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Independent Research DANSCATT has supported trips to the synchrotron source at ESRF, Grenoble, France. ABBREVIATIONS FEB, Free Energy Pertubation; JmjC, Jumonji C; KDM, Lysine demethylase; MD, Molecular Dynamics; PMTs, Posttranslational modifications; 2OG, Oxoglutarate.


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Biochemistry

REFERENCES [1] Kouzarides, T. (2007) Chromatin modifications and their function. Cell. 128(4), 693-705. [2] Mosammaparast, N., Shi, Y. (2010) Reversal of histone methylation: biochemical and molecular mechanisms of histone demethylases. Annu. Rev. Biochem. 79, 155-79. doi: 10.1146/annurev.biochem.78.070907.103946. [3] Allis, C. D., Berger, S. L., Cote, J., Dent, S., Jenuwien, T., Kouzarides, T., Pillus, L., Reinberg, D., Shi, Y., Shiekhattar, R., Shilatifard, A., Workman, J., Zhang, Y. (2007) New nomenclature for chromatin-modifying enzymes, Cell. 131(4), 633-6. [4] Kooistra, S. M., Helin, K. (2012) Molecular mechanisms and potential functions of histone demethylases. Nat. Rev. Mol. Cell Biol. 13(5), 297-311. doi: 10.1038/nrm3327. [5] Klose, R. J., Kallin, E. M., Zhang, Y. (2006) JmjC-domain-containing proteins and histone demethylation. Nat. Rev. Genet. 7(9), 715-27. doi: 10.1038/nrg1945. [6] Chi, P., Allis, C. D., Wang, G. G. (2010) Covalent histone modifications - miswritten, misinterpreted and mis-erased in human cancers. Nat. Rev. Cancer. 10(7), 457-69. doi: 10.1038/nrc2876. [7] Agger, K., Cloos, P. A. C., Christensen, J., Pasini, D., Rose, S., Rappsilber, J., Issaeva, I., Canaani, E., Salcini, A. E., Helin, K. (2007) UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature. 449(7163), 731-4. [8] Kruidenier, L., Chung, C., Cheng, Z., Liddle, J., Che, K., Joberty, G., Bantscheff, M., Bountra, C., Bridges, A., Diallo, H., Eberhard, D., Hutchinson, S., Jones, E., Katso, R., Leveridge, M., Mander, P. K., Mosley, J., Ramirez-Molina, C., Rowland, P., Schofield, C. J.,


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Sheppard, R. J., Smith, J. E., Swales, C., Tanner, R., Thomas, P., Tumber, A., Drewes, G., Oppermann, U., Patel, D. J., Lee, K. & Wilson, D. M., (2012) A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature. 488(7411), 404-8. doi: 10.1038/nature11262. [9] Walport, L. J., Hopkinson, R. J., Vollmar, M., Madden, S. K., Gileadi, C., Oppermann, U., Schofield, C. J. & Johansson, C. (2014) Human UTY(KDM6C) is a male-specific Nϵ-methyl lysyl demethylase. J. Biol. Chem. 289(26), 18302-13. doi: 10.1074/jbc.M114.555052. [10] Sengoku, T., Yokoyama, S. (2011) Structural basis for histone H3 Lys 27 demethylation by UTX/KDM6A. Genes. Dev. 25(21), 2266-77. doi: 10.1101/gad.172296.111. [11] Kristensen, B. L., Nielsen, A. L., Jørgensen, L., Kristensen, L. H., Helgstrand, C., Juknaite, L., Kristensen, J. L., Kastrup, J. S., Clausen, R. P., Olsen, L., Gajhede, M. (2011) Enzyme kinetic studies of histone demethylases KDM4C and KDM6A: towards understanding selectivity of inhibitors targeting oncogenic histone demethylases. FEBS Lett. 585(12), 1951-6. doi: 10.1016/j.febslet.2011.05.023. [12] Jacobson, M. P., Pincus, D. L., Rapp, C. S., Day, T. J., Honig, B., Shaw, D. E., Friesner, R. A. (2004) A hierarchical approach to all-atom protein loop prediction. Proteins. 55(2), 351-67. [13] Desmond Molecular Dynamic System, version 3.8 (2014) D. E. Shaw Research, New York , NY. [14] Guerois, R., Nielsen, J. E., Serrano, L. (2002) Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations. J. Mol. Biol. 320(2), 36987.


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[15] Humphrey, W., Dalke, A., Schulten, K. (1996) VMD: visual molecular dynamics. J. Mol. Graph. 14(1), 33-8, 27-8. [16] Dimasi, N., Flot, D., Dupeux, F., Márquez, J. A. (2007) Expression, crystallization and Xray data collection from microcrystals of the extracellular domain of the human inhibitory receptor expressed on myeloid cells IREM-1. Acta Crystallogr. Sect F. 63(Pt 3), 204-8. [17] Nurizzo, D., Mairs, T., Guijarro, M., Rey, V., Meyer, J., Fajardo, P., Chavanne, J., Biasci, J. C., McSweeney, S., and Mitchell, E., (2006) The ID23-1 structural biology beamline at the ESRF. J. Synchrotron Radiat. 13(Pt 3), 227-38. [18] Kabsch, W. (2010) XDS. Acta Crystallogr. D Biol. Crystallogr. 66(Pt 2),125-32. doi: 10.1107/S0907444909047337. [19] Evans, P. R. (2011) An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D Biol Crystallogr. 67(Pt 4), 282-92. doi: 10.1107/S090744491003982X. [20] McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., Read, R. J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40(Pt 4), 658-674. [21] Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., McCoy, A.J., Moriarty, N.W. , Oeffner, R., Read, R.J., Richardson, D.C., Richardson, J.S., Terwilliger, T.C., Zwart, P.H. (2010) Acta Crystallogr. D Biol Crystallogr. 66(Pt 2), 213-21. doi: 10.1107/S0907444909052925.


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Figure 1. ccKDM6B dose-response curves for H3(17-33) peptides. Data points were produced using a MALDI-TOF activity assay. 176x76mm (300 x 300 DPI)


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Biochemistry

Figure 2. Alignment of backbone carbon in catalytic domains of ccKDM6B:H3(17-33)A21M complex (green) and (a) human catalytic core KDM6A:H3(17-33) complex (pink, PDB code 3AVR[10]) RMS 0.69Å, (b) human catalytic core KDM6B (yellow, PDB code 2XUE [8]) RMS 0.57Å and (c) mouse catalytic core KDM6B:H3(2434) complex (blue, PDB code 4EZH [8]) RMS 0.42Å. 83x190mm (300 x 300 DPI)


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3: The L20 hydrophobic binding pocket (a) ccKDM6B (green) and (b) to catalytic core KDM6A (pink). 49x90mm (300 x 300 DPI)


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Biochemistry

Figure 4: Binding of H3(17-33)A21M peptide residue M21 to ccKDM6B (green). Neighbouring molecule in crystal at interface by H3M21 (wheat). 83x83mm (300 x 300 DPI)


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5: Binding of H3(17-33) peptide residue K23 to (a) ccKDM6B, clear electron is seen for the side chain of K23 indicating a fixed position, and (b) to catalytic core KDM6A, election density is not clear for the side chain of K23, indicating flexibility of this side chain. 83x160mm (300 x 300 DPI)


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Page 35 of 36 Biochemistry 1 2 3 4 5 ACS Paragon Plus Environment 6 7

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For Table of Contents use only 59x47mm (300 x 300 DPI)


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Structural Basis of Histone Demethylase KDM6B Histone 3 Lysine 27

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