KDM - media.nature.com · istone pep d with pep eptides. tides as tide only . Supplem MALDI-TO by recom black. entary Fig F MS of d binant KDM ure 10 K emethylati 3A. React DM3A doe

SupplemdemethylaReactions

SupplemdemethylaReactions

mentary Figation of thes containing

mentary Figation of ths containing

gure 1 KDMe shown vg enzyme a

gure 2 PHFhe shown vg enzyme a

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F8 catalysvariant histre in red wi

yses only lone peptideth peptide o

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ysine demes as catalonly control

ysine demedes as cataonly control

methylation.ysed by res in black.

ethylation. alysed by s in black.

. MALDI-TOecombinant

MALDI-TOrecombina

OF MS of KDM2A.

OF MS of nt PHF8.

SupplemMS of deKDM3A. text for ot

SupplemMS of deKDM4E. text for ot

mentary FigemethylatioReactions cther KDM3A

mentary FigemethylatioReactions cther KDM4E

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Fig. 2b).

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Fig. 2b).

ses lysine aant histonee in red with

ses lysine aant histonee in red with

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SupplemMS of deKDM5C. text for ot

SupplemMS of deKDM6B.

mentary FigemethylatioReactions cther KDM5C

mentary FigemethylatioReactions

ure 5 KDMon of the scontaining eC assays (F

ure 6 KDMon of the s

containing

M5C catalysshown varienzyme are

Fig. 2b).

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ses lysine aant histonee in red wit

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Supplementary Table 1 Peptide sequences used in the biochemical assays. All peptides were prepared as C-terminal amides. In some cases small shifts from these masses are observed in the MS due to shifts in the calibration. In all cases demethylation is observed as a shift of 14 Da from the substrate peak. Histone Mark Amino Acid Sequence Monoisotopic

Mass / Da Enzyme for which activity is characterised

H3K4me3 ART-Kme3-QTARKSTGGKA 1602 KDM5C H3K4Rme2a ART-Rme2a-QTARKSTGGKA 1615 H3K4Rme2s ART-Rme2s-QTARKSTGGKA 1615 H3K4Rme ART-Rme-QTARKSTGGKA 1601 H3K9me3 ARTKQTAR-Kme3-STGGKA 1602 KDM4A/E H3K9me2 ARTKQTAR-Kme2-STGGKA 1587 KDM3A H3K9Rme2a ARTKQTAR-Rme2a-STGGKA 1615 H3K9Rme2s ARTKQTAR-Rme2s-STGGKA 1615 H3K9Rme ARTKQTAR-Rme-STGGKA 1601 H3K4me3K9me2 ART-Kme3-QTAR-Kme2-STGGKA 1631 KDM7B H3K4me3K9Rme2a ART-Kme3-QTAR-Rme2a-STGGKA 1658 H3K4me3K9Rme2s ART-Kme3-QTAR-Rme2s-STGGKA 1658 H3K4me3K9Rme ART-Kme3-QTAR-Rme-STGGKA 1644 H3K27me3 KQLATKAAR-Kme3-SAPSTG 1656 KDM6B H3K27Rme2a KQLATKAAR-Rme2a-SAPAT 1596 H3K27Rme2s KQLATKAAR-Rme2s-SAPAT 1596 H3K27Rme KQLATKAAR-Rme-SAPAT 1582 H3K27me3 (21mer) KAPRKQLATKAAR-Kme3-

SAPATGG 2150 KDM6B

H3K27Rme2a (21mer)

KAPRKQLATKAAR-Rme2a-SAPATGG

2163

H3K36me2 APATGGV-Kme2-KPHRYRP 1661 KDM2A H3K36Rme2a APATGGV-Rme2a-KPHRYRP 1689 H3K36Rme2s APATGGV-Rme2s-KPHRYRP 1689 H3K36Rme APATGGV-Rme-KPHRYRP 1675 H3R2me2a A-Rme2a-TKQTARKSTGGKA 1587 H3R2me2s A-Rme2a-TKQTARKSTGGKA 1587 H3R2me A-Rme-TKQTARKSTGGKA 1573 H3R2Kme3 A-Kme3-TKQTARKSTGGKA 1575 H3R8me2a ARTKQTA-Rme2a-KSTGGKA 1587 H3R8me2s ARTKQTA-Rme2s-KSTGGKA 1587 H3R8me ARTKQTA-Rme-KSTGGKA 1573 H3R17me2a STGGKAP-Rme2a-KQLATKA 1540 H3R17me2s STGGKAP-Rme2a-KQLATKA 1540 H3R17me STGGKAP-Rme-KQLATKA 1526 H3R26me2a KQLATKAA-Rme2a-KSAPAT 1568 H3R26me2s KQLATKAA-Rme2a-KSAPAT 1568 H3R26me KQLATKAA-Rme-KSAPAT 1554 H4R3me2a (15mer) SG-Rme2a-GKGGKGLGKGGA 1314 H4R3me2s (15mer) SG-Rme2s-GKGGKGLGKGGA 1314 H4R3me2a (16mer) SG-Rme2a-GKGGKGLGKGGAK 1441 H4R3me2s (16mer) SG-Rme2s-GKGGKGLGKGGAK 1441 H4R3me SG-Rme-GKGGKGLGKGGAK 1427 53BP1(1394-1415) R1401me2a

GKAPVTP-Rme2a-GRGRRGRPPSRTTG

2345

53BP1(1394-1415) R1403me2a

GKAPVTPRG-Rme2a-GRRGRPPSRTTG

2345

53BP1(1394-1415) R1405me2a

GKAPVTPRGRG-Rme2a-RGRPPSRTTG

2345

BRCA1(605-619)R610me2a

APKKN-Rme2a-LRRKSSTRH 1862

hnRNPK(295-309)R296me2a

G-Rme2a-GGRGGSRARNLPL 1550

hnRNPK(295-309)R299me2a

GRGG-Rme2a-GGSRARNLPL 1550

FMR1(537-551)R544me2a

GGRGQGG-Rme2a-GRGGGFK 1430

FMR1(537-551)R546me2a

GGRGQGGRG-Rme2a-GGGFK 1430

p53(332-343)R333me2s

IRGRE-Rme2s-FEMFRE 1652

p53(328-339)R335me2s

FTLQI-Rme2s-GRERFE 1578

p53(330-341)R336me2s

LQIRG-Rme2s-ERFEMF 1608

Supplementary Table 2 Assay conditions with truncated recombinant protein.

Enzyme [2OG] / µM [Ascorbate] / µM [FeII] / µM Buffer Conditions

KDM2A 100 100 10 50 mM HEPES pH 7.5

KDM3A 100 100 50 50 mM HEPES pH 7.5

KDM4E 200 100 10 50 mM HEPES pH 7.5

KDM5C 100 100 10 50 mM HEPES pH 7.5

KDM6B 100 100 10 50 mM HEPES pH 7.5, 150 mM NaCl

PHF8 100 100 10 100 mM HEPES pH 7.5, 500 mM NaCl

Supplementary Table 3 Assay conditions with full length Flag-tagged KDMs.

Enzyme [2OG] / mM [Ascorbate] / mM [FeII] / µM Buffer Conditions

KDM3A 1 1 50 50 mM HEPES pH 7.5

KDM4A 1 1 50 50 mM HEPES pH 7.5

KDM5C 1 1 50 50 mM HEPES pH 7.5

KDM6B 1 1 50 50 mM HEPES pH 7.5, 150 mM NaCl

Supplementary Table 4. Crystallographic data processing and refinement statistics

PDB acquisition code 5FWE Data Collection Beamline (Wavelength, Å) I04‐1 (0.91741) Detector Pilatus 2M Data processing HKL20004 Space Group P21212 Cell dimensions a,b,c (Å)

100.739 149.400 57.372

No. of molecules/ ASU 2 Resolution (Å) 49.85 – 2.05 (2.12 – 2.05)* Completeness (%) 99.7 (99.8)* Redundancy 7.9 (8.1)* Rsym** 0.1 (1.0)*

Mean I/(I) 22.4 (2.5)*

Wilson B value (Å2) 33.8 Refinement Rwork / Rfree 0.234 / 0.256 No.reflections 54928(5334)*No.atoms ‐Enzyme(A/B) 2881/2850‐Metal(A/B) 2/2‐Ligand(A/B) 10/10‐Peptide(C/D) 17/29‐Water 392B‐factors ‐Enzyme(A/B) 47.9/47.5‐Metal(A/B) 32.8/30.4‐Ligand(A/B) 28.5/33.4‐Peptide(C/D) 73.3/74.1‐Water 45.9R.m.s. deviation Bond length, Å 0.007

Bond angle, 1.069

One crystal was used for structure determination.*Highest resolution shell shown in parenthesis. Polypeptide chain in parenthesis.

Supplementary References 1 Couture JF, Collazo E, Ortiz-Tello PA, Brunzelle JS, Trievel RC. Specificity and

mechanism of JMJD2A, a trimethyllysine-specific histone demethylase. Nature Structural and Molecular Biology 14, 689-695 (2007).

2 Chen Z, et al. Structural basis of the recognition of a methylated histone tail by JMJD2A.

Proc Natl Acad Sci U S A 104, 10818-10823 (2007).

3 Ng SS, et al. Crystal structures of histone demethylase JMJD2A reveal basis for

substrate specificity. Nature 448, 87-91 (2007). 4 Otwinowski, Z., Minor, W. Processing of X-ray Diffraction Data Collected in Oscillation

Mode. Methods in Enzymology 276, 307-326 (1997).

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KDM - media.nature.com · istone pep d with pep eptides. tides as tide only . Supplem MALDI-TO by recom black. entary Fig F MS of d binant KDM ure 10 K emethylati 3A. React DM3A doe