Supplementary Materials and Methods

Crystal structure determination

Diffraction data were collected at rotating anode or synchrotron radiation sources and

processed with XDS (Kabsch 2010) and AIMLESS (Evans and Murshudov 2013). The

SPKRIA complex structure was solved by molecular replacement using PHASER

(McCoy et al. 2007) with coordinates of PDB entry 2EX4, and subsequently served as

starting model for crystal structures of the other peptides’ complexes. Programs COOT

(Emsley et al. 2010), REFMAC (Murshudov et al. 2011), MOLPROBITY (Chen et al.

2010) and the PARVATI server (Merritt 1999) were used for rebuilding, restrained

refinement, geometry validation and model anisotropy analysis, respectively. Geometry

restraints for methylated proline were calculated with PRODRG (Schuttelkopf and van

Aalten 2004). IOTBX software (Gildea et al. 2011), PDB_EXTRACT (Yang et al. 2004)

as well as CCP4 (Winn et al. 2011) and PHENIX (Adams et al. 2010) programs were

used to compile model statistics.

Mass spectrometry

MALDI-MS based methylation assay was carried out through Applied Biosystems

Voyager matrix-assisted laser desorption/ionization time-of-flight mass spectrometer to

monitor the methylation of peptides in the presence of NTMT1 without addition of SAM

(Richardson et al. 2015). The assay was set up as the ITC condition. At 2h, sample was

withdrawn from reaction mixtures and 1:1 quenched with 3µL of quenching solution

(50% MeCN/20mM ammonium phosphate/0.4%TFA). 1µL of each samples were spotted

with 1µL � -Cyano-3-hydroxycinnamic acid matrix solution, and analyzed by

Biosystems Voyager matrix-assisted laser desorption/ionization time-of-flight mass

spectrometer. Obtained data was processed through Data Explore TM.

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McCoyAJ,Grosse-KunstleveRW,AdamsPD,WinnMD,StoroniLC,ReadRJ.2007.

Phasercrystallographicsoftware.Journalofappliedcrystallography40:658-674.

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proteinstructurerefinement.ActacrystallographicaSectionD,Biologicalcrystallography55:1109-1117.

MurshudovGN,SkubakP,LebedevAA,PannuNS,SteinerRA,NichollsRA,WinnMD,

LongF,VaginAA.2011.REFMAC5fortherefinementofmacromolecular

crystalstructures.ActacrystallographicaSectionD,Biologicalcrystallography67:355-367.

RichardsonSL,MaoY,ZhangG,HanjraP,PetersonDL,HuangR.2015.Kinetic

mechanismofproteinN-terminalmethyltransferase1.TheJournalofbiologicalchemistry290:11601-11610.

SchuttelkopfAW,vanAaltenDM.2004.PRODRG:atoolforhigh-throughput

crystallographyofprotein-ligandcomplexes.ActacrystallographicaSectionD,Biologicalcrystallography60:1355-1363.

WinnMD,BallardCC,CowtanKD,DodsonEJ,EmsleyP,EvansPR,KeeganRM,

KrissinelEB,LeslieAG,McCoyAetal.2011.OverviewoftheCCP4suiteand

currentdevelopments.ActacrystallographicaSectionD,Biologicalcrystallography67:235-242.

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andaccuratedepositionofstructuressolvedbyX-raydiffractiontothe

ProteinDataBank.ActacrystallographicaSectionD,Biologicalcrystallography60:1833-1839.

Supplementary Table 1 Data collection and refinement statistics

PDB$code$ 5E1B$ 5E1M$ 5E1O$ 5E1D$ 5E2B$ 5E2A$peptide$sequence$

SPKRIA$ PPKRIA$ RPKRIA$ YPKRIA$ (P0M)PKRIA$ (LSE)SPKRIA$

Data$collection$and$reduction$

$ $ $ $ $ $

Radiation$source$ APS$BEAMLINE$19EID$

Rigaku$FREE$ APS$BEAMLINE$24EIDEE$

APS$BEAMLINE$24EIDEE$

Rigaku$FREE$ rigaku$frEe$

Radiation$wavelength$[A]$

0.9793$ 1.5418$ 0.9792$ 0.9792$ 1.5418$ 1.5418$

Space$group$ P$65$2$2$ P$65$2$2$ P$65$2$2$ P$65$2$2$ P$65$2$2$ P$65$2$2$Cell$dimensions$a,b,c$[A],alpha,beta,gamma$[deg]$

107.3,107.3,205.4,90.00,90.00,120.00$

107.4,107.4,205.6,90.00,90.00,120.00$

107.5,107.5,206.4,90.00,90.00,120.00$

107.1,107.1,206.0,90.00,90.00,120.00$

107.3,107.3,205.6,90.00,90.00,120.00$

107.2,107.2,205.6,90.00,90.00,120.00$

Resolution$limits$[A]$

47.54E1.60(1.63E1.60)$

29.68E1.75(1.78E1.75)$

84.83E2.00(2.05E2.00)$

29.61E1.45(1.47E1.45)$

29.65E1.80(1.84E1.80)$

29.62E1.75(1.78E1.75)$

Unique$HKLs$ 92380(4488)$ 71201(3848)$ 48256(3461)$ 122141(5867)$

64736(3713)$ 70896(3828)$

Completeness$[%]$

100.0(100.0)$ 100.0(99.9)$ 99.9(99.7)$ 99.3(98.3)$ 99.2(98.0)$ 100.0(99.9)$

Rsym$ 0.134(1.213)$ 0.108(0.942)$ 0.171(0.880)$ 0.095(1.249)$ 0.130(1.180)$ 0.125(1.139)$I/sigma$ 21.7(3.2)$ 27.0(4.3)$ 23.0(5.0)$ 25.1(3.1)$ 27.7(3.6)$ 27.6(3.5)$Redundancy$ 21.7(21.8)$ 20.6(20.4)$ 21.5(22.1)$ 21.7(22.0)$ 21.1(21.1)$ 20.8(19.8)$Model$refinement$

$ $ $ $ $ $

Refinement$resolution$[A]$

50.01E1.65$ 50.01E1.75$ 84.81E2.00$ 30.00E1.45$ 29.65E1.95$ 29.60E1.75$

Reflections$used/free$

80997/3324$ 68505/2625$ 46342/1857$ 117420/4677$

49282/2019$ 68176/2626$

Number$of$atoms/average$BEfactor$[A**2]$

4368/17.5$ 4308/19.2$ 4221/21.9$ 4372/17.3$ 4288/19.3$ 4297/18.1$

$$Protein$ 3691/16.4$ 3673/18.3$ 3638/21.1$ 3741/16.4$ 3672/18.6$ 3657/17.2$$$Peptide$ 111/21.9$ 105/23.1$ 96/32.6$ 109/19.5$ 116/22.2$ 104/21.3$$$Water$ 436/25.0$ 417/26.0$ 387/28.3$ 389/25.3$ 385/24.8$ 426/24.8$$$Others$ 130/18.3$ 113/18.2$ 100/19.0$ 133/17.6$ 115/18.9$ 110/17.9$R$work/free$ 0.151/0.176$ 0.161/0.188$ 0.151/0.189$ 0.154/0.169$ 0.152/0.188$ 0.157/0.184$RMSD$bonds$[A]/angles$[deg]$

0.018/1.8$ 0.017/1.8$ 0.019/1.8$ 0.017/1.8$ 0.017/1.8$ 0.016/1.8$

Molprobity$Ramachandran$favored/outliers$[%]$

98.76/0.00$ 98.74/0.00$ 98.51/0.00$ 98.58/0.00$ 98.74/0.00$ 98.75/0.21$

$

Fig.%S1%Detailed(interac,ons(between(the(N3terminal(four(residues(of(the(RCC1(pep,de((purple(s,cks)(and(NTMT1((orange(s,cks(or(arcs).(Hydrophobic(interac,ons(of(NTMT1(residues(are(represented(by(orange(arcs,(hydrogen(bond(partners(by(orange(s,cks.(

A

C

B

D

Fig.%S2%%The(substrate(pep,des(bind(in(a(conserved(mode(to(NTMT1.(A:(Superposi,on(of(different(substrates((S/P/Y/RPKRIA)(in( the( electrosta,c( poten,al( surface( of(NTMT1.( The( first( residue( is( shown( in( s,ck(mode( and( colored( in( yellow,( orange,(magenta(and(cyan,(respec,vely.(SAH(is(shown(as(green(s,cks.(B3D:(Close3up(view(of(substrate(binding((P/Y/RPKRIA()(in(the(ac,ve(site.(In(addi,on(to(common(interac,ons,(P1(forms(hydrophobic(interac,on(with(W20((B),(Y1(and(R1(form(hydrogen3bonds(with(D180((C,(D).(

A B

Fig.% S3% Crystal( structures( of( NTMT13SAH3me13SPKRIA( and( NTMT13SAH3me13PPKRIA.( A:( Overlay( of( the(ac,ve(sites(of(NTMT13SAH3SPKRIA(and(NTMT13SAH3me13SPKRIA(complexes.(B:(Overlay(of(the(ac,ve(sites(of( NTMT13SAH3PPKRIA( and( NTMT13SAH3me13PPKRIA( complexes.( As( the( backbone( atoms( of( SAH( and(NTMT1(are(barely(changed,(only(the(substrates(are(overlaid(for(clarity.((

Fig.%S4%%Kine,c(analysis(of(NTMT1(with(different(pep,des(

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

-8.00

-4.00

0.00

-3.00

-2.00

-1.00

0.00

-10 0 10 20 30 40 50 60 70 80 90 100110120130

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

0.0 0.5 1.0 1.5 2.0 2.5 3.0

-16.00

-12.00

-8.00

-4.00

0.00

-2.00

-1.00

0.00

-10 0 10 20 30 40 50 60 70 80 90 100110120130

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0-3.00

-2.00

-1.00

0.00

-1.00

-0.80

-0.60

-0.40

-0.20

0.00

-10 0 10 20 30 40 50 60 70 80 90 100110120130

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0-12.00

-9.00

-6.00

-3.00

0.00-0.40

-0.30

-0.20

-0.10

0.00

-10 0 10 20 30 40 50 60 70 80 90 100 110

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

0.0 0.5 1.0 1.5 2.0

-12.00

-9.00

-6.00

-3.00

0.00

-2.00

-1.50

-1.00

-0.50

0.00

-10 0 10 20 30 40 50 60 70 80 90 100110120130

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

YPKRIA

Kd1=4.1µM

Kd2=0.1µM

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

-12.00

-9.00

-6.00

-3.00

0.00

-2.00

-1.50

-1.00

-0.50

0.00

-10 0 10 20 30 40 50 60 70 80 90 100 110

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0-12.00

-9.00

-6.00

-3.00

0.00

-0.80

-0.60

-0.40

-0.20

0.00

-10 0 10 20 30 40 50 60 70 80 90 100 110

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.012.00

9.00

6.00

3.00

0.00

-0.40

-0.20

0.00

-10 0 10 20 30 40 50 60 70 80 90 100 110

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

LPKRIA

Kd=48µM

DPKRIA NB

SIKRIA

NB

SEKRIA

NB

SPKRIA Kd1=14µM Kd2=0.8µM

SSKRIA

NB

WPKRIA

Kd=47µM

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

-9.00

-6.00

-3.00

0.00

-0.60

-0.40

-0.20

0.00

-10 0 10 20 30 40 50 60 70 80 90 100 110

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

SQKRIA

NB

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

-15.00

-10.00

-5.00

0.00

-2.00

-1.00

0.00

-10 0 10 20 30 40 50 60 70 80 90 100 110

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

RPKRIA

Kd1=3.7µM

Kd2=0.3µM 0.0 0.5 1.0 1.5

-30.00

-20.00

-10.00

0.00

-1.50

-1.00

-0.50

0.00

-10 0 10 20 30 40 50 60 70 80 90 100110120130

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

PPKRIA

Kd1=189nM Kd2=2.8nM

Fig.%S5%%ITC(,tra,on(curves(of(NTMT1(with(different(pep,des(

PPKRIA + NTMT1 PPKRIA [M+H]+

[M+Me+H]+

[M+Na]+

[M+2Me]+

[M+H]+

[M+Na]+

[M+H]+

[M+Me+H]+

[M+Na]+

[M+H]+

[M+Na]+

SPKRIA + NTMT1 SPKRIA

Fig.%S6A%%MALDI(spectra(of((S/P)PKRIA(in(the(presence(or(absence(of(NTMT1((

[M+H]+

[M+Me+H]+

[M+Na]+

[M+H]+

[M+Na]+

YPKRIA + NTMT1 YPKRIA

[M+H]+

[M+Me+H]+

[M+Na]+

[M+H]+

[M+Na]+

RPKRIA + NTMT1 RPKRIA

Fig.%S6B%%MALDI(spectra(of((Y/R)PKRIA(in(the(presence(or(absence(of(NTMT1((

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5-4.00

-2.00

0.00

-1.00

-0.80

-0.60

-0.40

-0.20

0.00

-10 0 10 20 30 40 50 60 70 80 90 100 110

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5-4.00

-2.00

0.00

-1.00

-0.80

-0.60

-0.40

-0.20

0.00

-10 0 10 20 30 40 50 60 70 80 90 100 110

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

0.0 0.5 1.0 1.5 2.0 2.5 3.0

-16.00

-12.00

-8.00

-4.00

0.00

-2.00

-1.00

0.00

-10 0 10 20 30 40 50 60 70 80 90 100110120130

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

NTMT1-WT

Kd1=14µM Kd2=0.8µM

NTMT1-D180A

NB

NTMT1-D180K

NB

Fig.%S7%%ITC(,tra,on(curves(of(NTMT1(and(its(mutants(with(hRCC1(pep,de((SPKRIA)(

0.0 0.5 1.0 1.5 2.0-4.00

-2.00

0.00

-0.60

-0.40

-0.20

0.00

-10 0 10 20 30 40 50 60 70 80 90 100110120130

Time (min)

µcal

/sec

Molar Ratio

KC

al/M

ole

of In

ject

ant

NTMT1-D177A

Kd=32µM

A

C

B

Fig.% S8% % %A( conserved( cataly,c( mechanism( for( different(substrates( (A3C,( P/Y/RPK)( of( NTMT1.( The( cataly,c( site( is(illustrated( in( the( same( way( as( in( Fig( 3D.( In( the( crystal(structures,(some(instances(of(W1(and(W2(are(occupied(by(glycerol( or( other( co3solutes.( For( discussion( of( the(proposed( mechanism,( we( labeled( the( co3solute( sites( as(waters.((