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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
M2A catalyariant histore in red wi
F8 catalysvariant histre in red wi
yses only lone peptideth peptide o
es only lytone peptidth peptide o
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
ure 3 KDMon of the scontaining eA assays (F
ure 4 KDMon of the scontaining eE assays (F
M3A catalysshown varienzyme are
Fig. 2b).
M4E catalysshown varienzyme are
Fig. 2b).
ses lysine aant histonee in red with
ses lysine aant histonee in red with
and arginine peptides h peptide o
and arginine peptides h peptide o
ne demethyas catalys
only controls
ne demethyas catalys
only controls
ylation. MAsed by recs in black. S
ylation. MAsed by recs in black. S
ALDI-TOF combinant See main
ALDI-TOF combinant See main
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).
M6B catalysshown vari enzyme a
ses lysine aant histonee in red wit
ses lysine aant histoneare in red
and arginine peptides h peptide o
and arginine peptides
with pepti
ne demethyas catalys
only controls
ne demethyas catalys
de only co
ylation. MAsed by recs in black. S
ylation. MAsed by recontrols in b
ALDI-TOF combinant See main
ALDI-TOF combinant black. (a)
reactions reaction w
SupplemNMR spe(residuesand the aare highH3K27Rmcorresponhighlighteshowing KDM6B-csequence
with 15mwith 21mer
mentary Figectrum of 18-32, se
arginine methlighted. (bme2a peptinding to theed. The formtime-depen
catalysed de KAPRKQL
er argininepeptide. Se
ure 7 NMRKDM6B-ca
equence KQthyl groups
b) 1H-13C-Hide (residue methyl gromaldehyde ndent formademethylatioLATKAARR
e methylateee main text
R analysis oatalysed deQLATKAARof substrate
HSQC spees 18-32, oup of Nω-mscavenger
ation of Non of a 2
Rme2aSAPA
ed-peptidest for other K
of KDM6B emethylationRRme2aSAPe and mono
ectrum of sequence
monomomer dimedone
Nω-monomom21 residueATGG).
(16mer lyKDM6B ass
catalysed an of a 15 PAT). 1H-reomethylatedKDM6B-caKQLATKA
thylarginine is presentmethylarginH3K27Rm
ysine methyays (Fig. 2b
arginine deresidue H
esonances d product peatalysed deAARRme2aSe (δH 2.76 pt in the samnine and sue2a peptid
ylated-peptb).
emethylatioH3K27me2a
for 2OG, eptide (δH 2emethylatioSAPAT). T
ppm, δC 27.mple. (c) 1Huccinate d
de (residue
tide) (b)
on. (a) 1H a peptide succinate
2.76 ppm) n of an
The peak 5 ppm) is H spectra uring the
es 14-34,
SupplemMALDI-TOcatalysedcontrols in
mentary FigOF MS of
d by recombn black See
gure 8 KDMf demethylabinant KDMe main text f
M4E catalyation of thM4E. Reactfor other as
yses argine shown ations contaissays (Fig. 4
ine demetharginine mening enzym4a).
hylation inethylated h
me are in re
n histone phistone peped with pep
peptides. ptides as ptide only
SupplemMALDI-TOcatalysedcontrols in
mentary FigOF MS of
d by recombn black See
gure 9 KDMf demethylabinant KDMe main text f
M5C catalyation of thM5C. Reactfor other as
yses argine shown ations contaissays (Fig. 4
ine demetharginine mening enzym4b).
hylation inethylated h
me are in re
n histone phistone peped with pep
peptides. ptides as ptide only
SupplemMALDI-TOby recomblack.
mentary FigOF MS of d
mbinant KDM
gure 10 KdemethylatiM3A. React
DM3A doeon of the stions contai
es not cathown arginining enzym
talyse histine methyla
me are in re
tone arginated histoneed with pep
ine demete peptides ptide only c
thylation. catalysed
controls in
SupplemMALDI-TOby recomblack.
mentary FigOF MS of d
mbinant KDM
gure 11 KdemethylatiM6B. React
DM6B doeon of the stions contai
es not cathown arginining enzym
talyse histine methyla
me are in re
tone arginated histoneed with pep
ine demete peptides ptide only c
thylation. catalysed
controls in
Supplemblot analyKDMs webead demdetected
SupplemMALDI-TOKDM3A impeptide o
mentary Figysis of HEKere immunomethylationusing anti-F
mentary FigOF MS of mmunoprecnly controls
gure 12 OveK293T cell precipitated assays (F
Flag primary
ure 13 Fulldemethylat
cipitated fros in black. S
erexpressiolysates exo
d using antiFig. 5, Supy antibody (
l length KDtion of the om HEK293See main tex
on of full logenously -flag beadspplementary(for source
DM3A catalshown his
3T cells. Rext for other
length KDMexpressing
s to producey Fig. 13-1see Method
lyses argintone peptid
eactions conassays (Fig
Ms in HEK2full length
e full length 16). Full leds).
nine and lysdes as catantaining enzg. 5).
293T cells.Flag-taggeKDMs for u
ength prote
ysine demealysed by fzyme are in
. Western ed KDMs. use in on-eins were
thylation. full length n red with
SupplemMALDI-TOKDM4A impeptide o
mentary FigOF MS of mmunoprecnly controls
ure 14 Fulldemethylat
cipitated fros in black. S
l length KDtion of the om HEK293See main tex
DM4A catalshown his
3T cells. Rext for other
lyses argintone peptid
eactions conassays (Fig
nine and lysdes as catantaining enzg. 5).
ysine demealysed by fzyme are in
thylation.
full length n red with
SupplemMALDI-TOKDM5C ipeptide o
SupplemMALDI-TOKDM6B impeptide o
mentary FigOF MS of mmunoprecnly controls
mentary FigOF MS of mmunoprecnly controls
ure 15 Fulldemethylat
cipitated fros in black. S
ure 16 Fulldemethylat
cipitated fros in black. S
l length KDtion of the om HEK293See main tex
l length KDtion of the om HEK293See main tex
DM5C catalshown his
3T cells. Rext for other
DM6B catalshown his
3T cells. Rext for other
lyses argintone peptid
eactions conassays (Fig
lyses argintone peptid
eactions conassays (Fig
nine and lysdes as catantaining enzg. 5).
nine and lysdes as catantaining enzg. 5).
ysine demealysed by fzyme are in
ysine demealysed by fzyme are in
thylation. full length n red with
thylation. full length n red with
SupplemMALDI-TOrecombinpeptide o
mentary FigOF MS ofant KDM5Cnly controls
ure 17 KDMf demethylC, KDM4Es in black.
Ms catalyslation of t, and KDM
e arginine he shown
M6B. React
demethylanon-histon
tions conta
ation in nonne peptideaining enzy
n-histone pes as catayme are in
peptides. alysed by
red with
Supplem
Stereovie
peptide re
asymmet
electron
indicating
suggested
KDM4A c
structure.
unproduc
conforma
the metal
Compare
chain wa
account fo
mentary Fig
ew from PD
esidues. KD
ric unit KDM
density wa
g lower occ
d binding o
chains A an
In KDM4
ctive since b
ations were
l to enable
d to methy
as observed
or their cata
ure 18 Ste
B 5FWE sh
DM4A.Ni.N
M4A (chains
as observed
cupancy (<1
of more tha
nd B; only t
A Chain A
both N-meth
refined in b
catalysis (w
ylated-lysine
d to adopt
alytic differe
reoview fro
howing the F
OG.H4R3m
s A and B).
d for resid
1.00) for su
an one con
the major c
A, the met
hyl groups a
both of whi
within 4.2 Å
e structures
more than
ences (Fig 6
om a KDM4
Fo-Fc OMIT
me2s comp
In both KD
ues in the
ubstrate re
nformations
conformatio
thylated arg
are too far f
ich one of t
Å) (b) as ob
s, it is of int
n one diffe
6).
4A.Ni.NOG
T map cont
lex crystalli
DM4A chains
H4R3me2
sidues. Th
of the me
ons were m
ginine conf
from the me
the N-meth
bserved for
terest that
rent extend
G.H4R3me2
toured to 3
ised with tw
s A and B, o
2s peptides
is unweight
thylated arg
odelled and
formation is
etal (>6.0 Å
yl groups w
N-methyl ly
the N-meth
ded conform
2s crystal s
around H4
wo molecul
only weak d
s (chains C
ted Fo-Fc O
rginine side
d refined in
s likely ca
Å) (a) In cha
was close e
ysine substr
hylated argi
mations, w
structure.
4R3me2s
es of per
difference
C and D)
OMIT map
echains in
n the final
atalytically
ain B, two
enough to
rates1, 2, 3.
nine side
hich may
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).