supplementary information crystal structure of …...supplementary information crystal structure of...

Supplementary information

Crystal structure of autotaxin and insight into

GPCR activation by lipid mediators

Hiroshi Nishimasu1, Shinichi Okudaira2, Kotaro Hama2, Emiko Mihara3, Naoshi

Dohmae4, Asuka Inoue2, Ryuichiro Ishitani1, Junichi Takagi3*, Junken Aoki2*,

Osamu Nureki1*

1Department of Biophysics and Biochemistry, Graduate School of Science, The University of

Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

2Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aoba, Aramaki, Aoba-ku,

Sendai, Miyagi 980-8578, Japan

3Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871,

Japan

4Biomolecular Characterization Team and CREST/JST, RIKEN, 2-1 Hirosawa, Wako,

Saitama 351-0198, Japan

*To whom reprint requests should be addressed:

Osamu Nureki ([email protected])

Junken Aoki ([email protected])

Junichi Takagi ([email protected])

Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988

Supplementary Discussion

Interdomain interaction

The catalytic domain extensively interacts with the two SMB-like domains on one side, and

with the nuclease-like domain on the other side (Fig. 1b,c). There is no contact between the

SMB-like and nuclease-like domains. The SMB-like domains 1 and 2 interact with the 9 and

11 helices and the 8–9 loop of the catalytic domain (Fig. 3a). Phe63, Asn77 and Leu94 of

the SMB-like domain 1 form van der Waals contacts with Ile279, Arg283, Thr287 and Trp291

of the catalytic domain, while Trp100 of the SMB-like domain 2 contacts Pro280, Glu282,

Arg283 and Leu286 of the catalytic domain. In addition, there are nine direct hydrogen bonds

between the catalytic domain and the SMB-like domains (Supplementary Table 1). The

nuclease-like domain interacts with the 15–12, 6–7 and 13–14 loops of the core

subdomain and the 12–13 region of the insertion subdomain of the catalytic domain (Fig. 3b).

The interactions between the two domains are mainly mediated through 15 direct hydrogen

bonds (Supplementary Table 1) and a water-mediated hydrogen bonding network involving

well-ordered water molecules located at the domain interface (Supplementary Fig. 7a,b). In

addition, there are van der Waals interactions (Pro429–His818, Arg202–Leu851,

His427–Arg794 and Lys430–His762) and a disulfide linkage (Cys413–Cys801). The

Asn524-linked glycan further reinforces the interdomain interaction, mainly through an

extensive hydrogen bonding network involving the bound waters (Fig. 1b,c and

Supplementary Fig. 7a).

The L1 linker interacts with the catalytic domain (Supplementary Fig. 7c), while the

long L2 linker interacts with both the catalytic and nuclease-like domains (Supplementary Fig.

7d). The Trp143 side chain of L1 is inserted into a hydrophobic pocket formed by Lys190,

Leu191, Met339 and Tyr490 of the catalytic domain. Cys156, Pro157, and Phe160 of L1 form a

hydrophobic core with Pro164, Leu292, Arg299, Leu347, Cys350 and Val351. The main-chain

NH and CO groups of Phe539 of L2 form bidentate hydrogen bonds with Asn531, with its side

chain stacked over His527 of the catalytic domain. The L1 and L2 linker regions form 15 and

31 direct hydrogen bonds, respectively, with the catalytic and nuclease-like domains

(Supplementary Table 1). In addition, the interactions between the linker regions and the

domains are reinforced by four disulfide linkages (Cys residues 148–194, 156–350, 566–662

and 568–647).


Supplementary Table

Supplementary Table 1 Interdomain hydrogen bonding interactions

Catalytic domain SMB-like domain 1

Residue Atom Residue Atom Distance (Å)

Thr272 OG1 Asn77 OD1 3.08

Phe274 O Asn77 ND1 2.83

Ser276 N Asn77 O 3.11

Trp291 NE1 Phe63 O 3.03

Catalytic domain SMB-like domain 2


Arg283 NH2 Lys95 O 2.93

Arg283 NH2 Ala116 O 2.90

Gln290 NE2 Arg126 O 2.77

Gln290 NE2 Gly127 O 3.25

Gln344 OE1 Gln134 N 2.76

Catalytic domain Nuclease-like domain


Tyr221 OH Arg821 NH2 3.13

Lys421 NZ Glu799 O 2.66

Tyr423 OH Glu799 OE2 2.73

Lys430 NZ Asp735 OD2 2.60

Arg431 NH2 Met817 O 3.16

Arg431 NH Met817 O 2.92

His433 NE2 Asp824 OD1 2.73

Arg449 NH1 Glu855 OE1 2.65

Arg449 NH2 Glu855 OE2 2.80

Thr526 OG1 Arg823 NE 2.96

Ser529 O Thr846 OG1 2.83

Asn531 ND2 Ser842 OG 2.94

Lys479 NZ Thr853 O 3.38

Lys479 NZ Glu855 OE1 3.42

Asn481 N Glu855 OE1 3.22

Catalytic domain L1 linker


Lys190 NZ Trp143 O 2.73

Lys190 NZ Asp146 O 2.86

Arg299 NH2 Gly159 O 3.01

Asp340 OD1 Trp143 N 2.90

Lys343 NZ Glu140 OE1 3.24

Lys343 NZ Ser141 OG 3.07

Lys343 NZ Ser141 O 2.70

Lys343 NZ Asp146 OD2 2.62

Arg349 NH1 Glu149 O 2.93


Arg349 NH2 Glu150 O 3.06

Arg349 NH2 Glu155 OE2 3.14

Arg349 NE Glu155 OE1 2.98

Arg349 O Glu155 N 2.91

Arg535 NH1 Glu150 OE1 3.11

Arg535 NH2 Glu150 OE1 2.95

Catalytic domain L2 linker


Asp514 O Asn537 ND2 3.35

Asn531 OD1 Phe539 N 2.96

Asn531 ND2 Phe539 O 2.64

Thr536 OG1 Asn537 ND2 2.69

Nuclease-like domain L2 linker


Leu546 O Cys662 N 2.94

Leu546 O Leu663 N 3.35

Thr587 N Lys584 O 2.98

Thr587 O Lys584 O 3.35

Arg596 NH2 Glu546 OE1 2.76

Arg596 NE Glu546 OE2 3.54

Val599 N Glu546 OE2 3.39

Val599 O Val547 N 2.92

Val599 O Ser548 N 3.37

Tyr601 O Ser548 OG 2.67

Thr603 N Ser548 OG 2.90

Thr603 O Ser548 OG 3.09

Tyr605 OH Glu546 OE2 3.32

Tyr605 O Asn551 N 2.96

Tyr605 O Asn551 ND2 3.08

Asp606 OD1 Asn551 ND2 3.14

Ile607 N Asn551 O 2.87

His643 NE2 Asp570 OD2 2.70

Val648 N Thr567 O 3.23

Arg649 NH2 Glu575 OE1 2.72

Val652 O Met556 N 3.16

Val652 O Tyr557 N 3.06

Glu686 O Asn572 ND2 3.24

Tyr689 OH Asp570 OD2 2.97

Asp690 OD1 His582 NE2 2.80

Arg835 NH1 Pro544 O 2.79

Arg835 NH2 Pro544 O 2.92


Extracellular

Cytosol

LPA

LPA receptor

Proliferation

Cellular response

MigrationSurvival

LPC

ATX

OP

OON+

O

OH

OO

OP

O O

O

OH

OO

–

–

–

Substrate

SMB-like domain 1 SMB-like domain 2

GPI-anchor

Enpp1 Nucleotide N C

C

C

C

C

C

C

LysophospholipidNucleotideATX/Enpp2 N

NucleotideEnpp3 N

NucleotideEnpp4 N

NucleotideEnpp5 N

LPCGlycerophosphocholineEnpp6 N

Enpp7 NLPCSphingomyelin

Enzyme

Catalytic domain Nuclease-like domain

a

b


1 10 20 30 40 50 60 70 80

M.musculus S C R E SD P GSCK RCFEL E P CRCDNLCK Y CC .......MARQGCFGSYQVI LFTFAIGVNL LGFTAS IKRAE.WD GPPTVL S WTNTS G Q VGP D S SS HH.sapiens S C R E SD P GSCK RCFEL E P CRCDNLCK Y CC MTRHADRMARRSSFQSCQII LFTFAVGVNI LGFTAH IKRAEGWE GPPTVL S WTNIS G Q AGP D S TS HX.laevis S C R E SD P GSCK RCFEL E P CRCDNLCK Y CC .......MAMKNGFSFHKVI LVTFAIGINV LGFTAN FKRSEEWD GVASVL S WIRSS E I AEA A S NS ED.rerio S C R E SD P GSCK RCFEL E P CRCDNLCK Y CC ............MLQLKWVF VMWLFSRLTV KTYVVR SGKAASPD SLSKSF Q FTSLA K V ADP N T NM S

90 100 110 120 130 140 150 160 170 180

M.musculus DFD CLKT G EC K RCGE RNE ACHCSEDC GDCCTNY CKG W CEE ECPAGFVRPP I SVDGFRASYMK G EL AR W T D V EN LSR QVV ESH VDDD IRVP L IF K SH.sapiens DFD CLKT G EC K RCGE RNE ACHCSEDC GDCCTNY CKG W CEE ECPAGFVRPP I SVDGFRASYMK G EL AR W T D V EN LAR QVV ESH VDDD IKAA L IF K SX.laevis DFD CLKT G EC K RCGE RNE ACHCSEDC GDCCTNY CKG W CEE ECPAGFVRPP I SVDGFRASYMK G EH GR W T D T EN LAK QVV GTH ADDD MKHP L IF K HD.rerio DFD CLKT G EC K RCGE RNE ACHCSEDC GDCCTNY CKG W CEE ECPAGFVRPP I SVDGFRASYMK G DH AG F S E N QH MAK RSL DVP LQEE IKNH V ML R G

190 200 210 220 230 240 250 260 270

M.musculus V PNI KLRSCGTH PYMRP YPTKT PNLYT TGLYPESHGIVGNS DP FDA F R EK NHRWWGGQP WITA KQG A FFW VK M E A V F LA MY V T HL GR F L T VR GT S H.sapiens V PNI KLRSCGTH PYMRP YPTKT PNLYT TGLYPESHGIVGNS DP FDA F R EK NHRWWGGQP WITA KQG A FFW VK M E S V F LA MY V T HL GR F L T VK GT S X.laevis V PNI KLRSCGTH PYMRP YPTKT PNLYT TGLYPESHGIVGNS DP FDA F R EK NHRWWGGQP WITA KQG A FFW VK M D S V F LA MY V N SL SR F I S LK AT P D.rerio

280 290 300 310 320 330 340 350 360 370

M.musculus I ERR T LQWL LPD ERP YA SEQ D GHK GP E L DK GQLM GLKQ KLHRC N I VGDHGME C TEFLS PH IL I S N SV FY P FS Y FGP MTNP REI TV D L V V F DVT DR H.sapiens I ERR T LQWL LPD ERP YA SEQ D GHK GP E L DK GQLM GLKQ KLHRC N I VGDHGME C TEFLV PH IL I T H SV FY P FS Y FGP MTNP REI IV D L V V F DVT DR X.laevis I ERR T LQWL LPD ERP YA SEQ D GHK GP E L DK GQLM GLKQ KLHRC N I VGDHGME C TEFLA SQ IF V H N YV LY P QA Y FQP LAEQ KVN IV D M V V F EAT ER D.rerio

380 390 400 410 420 430 440 450 460

M.musculus Y N D L PG GR R N K D KA ANLTCKKPDQHFKPY KQHLPKRLHYA N RIE HL V R WH ARK KSN LT VD IT V TL I PKIPN L Y P II M N R DL L E R V PLDVYKKPSG H.sapiens Y N D L PG GR R N K D KA ANLTCKKPDQHFKPY KQHLPKRLHYA N RIE HL V R WH ARK KSN LT VD IT V TL I SKFSN A Y P II L N R DI L E R V PLDVYKKPSG X.laevis Y N D L PG GR R N K D KA ANLTCKKPDQHFKPY KQHLPKRLHYA N RIE HL V R WH ARK KNS LS VD FA L SI M SRNPA . H P VV L F R DI L D K V PMDVYKR.QG D.rerio

470 480 490 500 510 520 530 540 550 560

M.musculus C F GDHG DNK SMQT F G GP FK TK PPFENIELYN MCD LGLKPA NNGTHGSLN LLR P P EV P F Q F VN V V Y T YR V V L P H TNTFR TL E SR NYPGIMYLQSDFH.sapiens C F GDHG DNK SMQT F G GP FK TK PPFENIELYN MCD LGLKPA NNGTHGSLN LLR P P EV P F Q F VN V V Y T YK V V L P H TNTFR TM E TR NYPGIMYLQSDFX.laevis C F GDHG DNK SMQT F G GP FK TK PPFENIELYN MCD LGLKPA NNGTHGSLN LLR P P EV P A Q Y IT V L H S YK V V V S H VASYK AI D SK LPIVTSPSTVNED.rerio C F GDHG DNK SMQT F G GP FK TK PPFENIELYN MCD LGLKPA NNGTHGSLN LLR P P EV P G A Y IN I L Y A FK I I L P Q TPVYI NM E TK NPAG.PVIAIND

570 580 590 600 610 620 630

M.musculus LGC C DKNK ELN RL L YGRPAV T Y L H D SG SE MPLWTSYT SKQ D T D LE K HTKGS..................TEERH L LYR S DI Y T FE Y IFL I AEVSSH.sapiens LGC C DKNK ELN RL L YGRPAV T Y L H D SG SE MPLWTSYT SKQ D T D LD K HTKGS..................TEERH L LYR R DI Y T FE Y IFL V AEVSSX.laevis LGC C DKNK ELN RL L YGRPAV T Y L H D SG SE MPLWTSYT SKQ E S D AE K YLKGTDDVAVEELSNEIKELTSRNTDKN L LYK K SV H S FE F SLM I ADVSGD.rerio LGC C DKNK ELN RL L YGRPAV T Y L H D SG SE MPLWTSYT SKQ D T E VD Q RQVID...................DNKN P MFR K CI H T YI Y ALH V VDFTP

640 650 660 670 680 690 700 710 720 730

M.musculus L C R D R SQ C Y KQ SY FL PP LSS YDA L TN P Y AFK W YFQ LVK A E NGVNV GPIFIPEH TN V P V VSPGF N LA KND M G F Y SPEAK F V MV M P RV T RV KY S R IS H.sapiens L C R D R SQ C Y KQ SY FL PP LSS YDA L TN P Y AFK W YFQ LVK A E NGVNV GPIFVPDH TS V P V VSPSF N LA KND M G F Y SPEAK F V MV M P RV N RV KY S R IS X.laevis L C R D R SQ C Y KQ SY FL PP LSS YDA L TN P Y AFK W YFQ LVK A E NGVNV GPIFIPEH SN V L P ISPGN S SA KAD M G F Q SADSK F I VI I P KI N RV RF T R IS D.rerio L C R D R SQ C Y KQ SY FL PP LSS YDA L TN P Y AFK W YFQ LVK A E NGVNV GPIFLTDA SN I P S VPTAY S SN RTE I S Y Q TQEAR V I TV M A RV S KS RY S K VT

740 750 760 770 780 790 800 810 820

M.musculus DY Y G D IK V G S PTHY TSC D Q D C G LSV SF PHR DN E CNSSE ESKWVE L K HT R RD E LT N N LR IEDE QY E S IPV YSII L FT PA K D P S IL P D S D E M M A V I H H.sapiens DY Y G D IK V G S PTHY TSC D Q D C G LSV SF PHR DN E CNSSE ESKWVE L K HT R RD E LT D D LH TEDK QY E S IPV YSII L FT PA K D P S IL P E S D E M M A V I H X.laevis DY Y G D IK V G S PTHY TSC D Q D C G LSV SF PHR DN E CNSSE ESKWVE L K HT R RD E LT D D VY TMDK MF D . IPV YYII M FN AV N D C V VI P D S E D L M T I I L D.rerio DY Y G D IK V G S PTHY TSC D Q D C G LSV SF PHR DN E CNSSE ESKWVE L K HT R RD E LT D N LR SAET QF S . VQI FVVV L YT TV S V P F IL S E T D E M T A L V L

830 840 850

M.musculus LDF R T R Y EIL LKTYLHTYESEI G Y K S S S T H.sapiens LDF R T R Y EIL LKTYLHTYESEI S F K S S P T X.laevis LDF R T R Y EIL LKTYLHTYESEI G Y K N S T S D.rerio LDF R T R Y EIL LKTYLHTYESEI G Y R S T E A

α1

α2 η1 α3 α4 η2 β1 η3

α5 β2 β3 α6 α7 β4 β5 η4 η5 α8

α9 β6 α10 η6 α11 β7 β8 β9

η7 η8 β10 β11 α12 β12 η9 η10 β13 β14 η11

β15 η12 β16 β17 β18 η13 α13 η14 η15

η16 α14 β19 β20 β21 β22

η17 η18 α15 β23 η19 η20 η21 β24 α16 α17 β25

η22 β26 β27 η23 α18 β28 α19

β29 α20

V PNI KLRSCGTH PYMRP YPTKT PNLYT TGLYPESHGIVGNS DP FDA F R EK NHRWWGGQP WITA KQG A FFW VT I E A M Y IT IH S N NF GK L I M VK GS P

I ERR T LQWL LPD ERP YA SEQ D GHK GP E L DK GQLM GLKQ KLHRC N I VGDHGME C TEFLA PM VL M H A YL MH L SY L HST LNSA RDV VI N M I I L EAH DK

Y N D L PG GR R N K D KA ANLTCKKPDQHFKPY KQHLPKRLHYA N RIE HL V R WH ARK KSS MS TE LI I SL I ARNPN S F A VV L N D EI M E K I IMKTKRN.HE

c


d

160 170 180 190 200 210 220 230 240 250

ATX/Enpp2 DGFR Y P G P TG E H N S TKT N YT LY GI MYD F AGFVRPPLIIF V AS MKKGS.KVM NIEKLRSC THAPYMRPVYP F L LA P S VG S PVFDAT HL.RGREKFNEnpp1 DGFR Y P G P TG E H N L S TKT NHY V LY GI MYD F AEFESPPT LF L AE LHTWG.GLL VISKLKNC TYTKNMRPMYP F SI P S ID K PKMNAS SL.KSKEKFNEnpp3 DGFR Y P G P TG E H N S TKT NHYT V LY GI MYD F PGFDLPPVILF M AE LQTWS.TLL NINKLKTC IHSKYMRAMYP F I P S ID N VHLNKN SL.SSVEKSNEnpp4 DGFR Y P G P TG E H N L S TKT NHY V LY GI MYD F SDSSAPRL LV F AD LKS...YDL HLQNFIKE VLVEHVKNVFI F SI E S VA S SVTKKH S..ESNDK.DEnpp5 DGFR Y P G P TG E H N L S TKT NHYT V L GI M D F LQPEEQKV VV F WD LYK...VPT HFHYIMKN VHVNQVTNVFI Y L FA N VA D F PILNKS SL.EHMDIYDEnpp6 DGFR Y P G P TG E H N L N YT M D F .ASAHRKL VLLL SD ISEDALASL GFREIVNR VKVDYLTPDFPSLSY Y LM RHC V QMIG Y W PRTNKS DIGVNRDSLMEnpp7 DGFR Y P G P TG E H N L S T T H T V Y G Y PVQRQHKL LV F WN DQDVD...T NLDSMAQE VKAQYMTPAFV M S C F L K I N VVH MF NTTSTVRLPY.HATLGIQ

... 260 270 280 290 300 310

ATX/Enpp2 P W T Y D GH GP WW G R WL E P HR G .Q L I ATK.QGVRAGTFFWS...................VS.IPHER ...ILTILQ SLPDNERPSVYAF S Q FS KY .Enpp1 P W T Y D GH GP W G I WPG V Y R WL EEP PL YK .Q V ANH.QEVKS.GT.YF SD EIDGILPDIYKV NGSVPFEE ...ILAVLE QLPSHERPHFYTL L SS SH .Enpp3 P W T Y D GH GP WW G I WPG V Y R WL EEP PA S .Q L AMY.QGLKA.AC.YY SD AVNGSFPTIYRN SNSVPYER ...ITTLLQ DLPKADRPSFYTI V SA SS .Enpp4 P W T Y D GH GP WW G I WPG V Y R WL EEP PF N AE V NQLQENRSS.AA.AM TD PIHNITASYFMN SSSVSFKE ...LGNVTT SSSNPP.VTFAAL W VS KY EEnpp5 P W T Y D GH GP W I WPG V Y R W EEP SKF EEAT I NQR.AGHAS.GA.AM AD KIHDSFPTYYLP NESVSFED ...VAKIIE FTAKDP..INLGFL W DT DV .Enpp6 P W T Y D GH GP WW G WPG V Y L E PL N SE L I LMK.ARRKV.YM.YY CE EILGVRPTYCLE KTVPTDINF...ANAVSDA DSLKSGRADLAAI H RI VE HY .Enpp7 P W T Y D GH GP W G I PG V EP RW DN SI I AQR.QGLKT.GS.FFY GN TYQGEAVTMSRKEGVLHNYKNETEWRGNVDTVMKWFLEEDVSLVTL FG ST KY .

320 330 340 350 360 370 380 390 400

ATX/Enpp2 D L N I DHGM L G L LK FGPEMTNP REI KTV Q MDG QLK HRCV V FVG ED....VTCDRTEFLSNYLTNVDDITLVPGTLGRIRPK.IPNN.LKYDPKAIIEnpp1 D L N I DHGM S L V G L LK S S V SEVIKA QK RLV M MDG DLG DKCL L LI EQ....G CKKYVYLNKYLGDVNNVKVVYGPAARLRPTDVPETYYSFNYEALAEnpp3 D L N I DHGM S L V G L LK S P V AGVIKA QS NAF M MEG QRN HNCV I VLA DQ....T CDRVEYMTDYFPKIN.FYMYQGPA RIRTRNIPQDFFTFNSEEIVEnpp4 D L N I DHGM L V G LK S S P DKENMRRV KE DLI DIVLK VLG WDSL V IT AQ....C .KNRLIDLDSCIDRSNYS.VIDLT VAAILPKIN.VTEVYD....Enpp5 D L N I DHGM S V G L LK S S P D PLMGSVISD HKL Y IKM RAK WNNV L VT TQ....C .KQRVIELDRYLDKEHYT.LIDHS VAAILPKEGKFDEVYD....Enpp6 D L N I DHGM S L V S P S PQRKDA RA TVLKYMIQWIQDRG QQDL V LF TD....IFWMDKVIELSNYISLDDLQQVKDRG VVSLWPVPGKHSEIYH....Enpp7 D L N I DHGM S V G L K S S P E QERKDMVKQ RTV Y RDSI RHH SDSL L IT TTVNKKA DLVEFHKFSNFTFQDIQFELLDYG IGMLIPKEGMLEKVYS....

.... 410 420 430 440 450 460 470 480 490

ATX/Enpp2 Y P G HG N M GP F H K R HY RI W D F ANLTCKKPDQ FKP M QHL K L ANNR EDLHLLVERR HVARKP....LDVYKKPSGKCFFQ D F KVNS QTV VGY T KYRTKEnpp1 Y P G HG N M GP F H K R H RI P D W D F KNLSCREPNQ FRP L PFL K L FAKSD E LTFYL PQ QLALN..........PSERKYCGS F S LFSN QAL IGY A KHGAEEnpp3 Y P G HG N M GP F H R HY RI D W F A RNLSCRKPDQ FKP LTPDL K L AKNV DKAHLMV RQ LAFRS..........KGSSNCG.G T YN EFKS EAI L H S IEKTVEnpp4 Y P G HG N M GP F H K R Y RI P W D A ...KLKRCNP MNV L EAI N FY QHSS Q IILVAEEG TITLN............KSS.FKL D Y SLPS HPFLA H A RKGYREnpp5 Y P G HG N M GP F K R HY R P D W D F A ...ALAGAHPNLTV K EEI E W KHND VQ IVAVA EG YILQN............KSDDFLL N Y ALAE HPI L H A RKNFTEnpp6 Y P G HG N M GP F H K R Y P D W D F A ...KLR.TVE MTV E ESI N FY KKGKFVS LTLVA EG FIAESREMLPFWMNSTGKREGWQR W Y ELMD RGI L I D KSNFREnpp7 Y P G HG N M GP F K HY RI P D F A ...VLKDAHPRLHV K EDF KNF ANNP T LLMYS LGYVIHGR............VNVQFNN E FN QDMD KTI R V S KAGLE

500 510 520 530 540 550 560 570 580

ATX/Enpp2 Y C P G L M LLG P NN L VPPFENIEL NV D LK A THGSLNH RTNTFRPTLPEEVSRPNYPGIMYLQSDFDLGCTCDDKNK....LEELNKRLHTKG....SEnpp1 Y C P G L M LLG P NN L VDSFENIEV NL D LI A SHGSLNH KKPIYNPSHPKEEGFLSQCPIKS..TSNDLGCTCDPWIVP...IKDFEKQLNLTTED.DDEnpp3 Y C P G L LL P NN L IEPFENIEV NLL D HIE A THGSLNH KTPFYKPSHAGELSTPADCGFTTPLPTDPLDCSCPALQNTPGLEEQANQRLNLSEGEVAAEnpp4 Y C P G L M LG P NN L QSTINTVDI PM HI LK H TLSHTKC VDQWCINLPEAIGIVVSALLVLTMLTGLMIFMRSRASTSRPFSRLQLQEDDDDPLIDVHTEnpp5 Y C P G L LL N L KEAMNSTDL SLL H NLTAL H SFWNVQD SSATPKPIPYTQSTTLLLGSDKPGEDEQEESYPYYIGVSLGSIIAMVFFVVLIKHLIRSQ Enpp6 Y C P G L M G P NN AAPIRSVDV NI HVA IT L SWSRVVCM KGQTSSAPPTPLNSCALVLILLLYFVEnpp7 Y C P G L M LLG P N VEPFESVHV EL Q IV E D NPGILRPM RSGSASLLSSQHHLVALLVGILTCLAKVL

590 600 610 620 630 640 650 660 670 680

ATX/Enpp2Enpp1 Enpp3

Enpp5 LQYRQVEVAQPLLQA

690 700 710 720 730 740 750 760 770

ATX/Enpp2Enpp1 Enpp3

780 790 800 810 820 830 840 850

ATX/Enpp2

Enpp3

β1 η3 α5 β2 β3 α6 α7 β4 β5 η4

η5 α8 α9 β6 α10

η6 α11 β7 β8 β9 η7 η8 β10 β11 α12

β12 η9 η10 β13 β14 η11 β15 η12 β16 β17

β18 η13 α14 η14 η15 η16

α14 β19 β20 β21 β22 η17 η18 α15 β23 η19

η20 η21 β24 α16 α17 β25 η22 β26

β27 η23 α18 β28 α19 β29 α20

1 10 20 30 40 50 60

ATX/Enpp2 .................................MARQGCFGSYQVISLFTFAIGVNLCLGFTASRIKRAEWDEGPPTVLSDSPWTNTSGSCKGRCEnpp1 MERDGDQAGHGPRHGSAGNGRELESPAAASLLAPMDLGEEPLEKAERARPAKDPNTYKVLSLVLSVCVLTTILGCIFGLKPS.CAKEVKSCKGRCEnpp3 MDSRLALATEEPIKKDSLKKYKILCVVLLALLVIVSLG.LGLGLGLRKP...EEQGSCRKKC

70 80 90 100 110 120 130 140 150

ATX/Enpp2 FELQEVGPPDCRCDNLCKSYSSCCHDFDELCLKTARGWECTKDRCGEVRNEENACHCSEDCLSRGDCCTNYQVVCKGESHWVDDDCEEIRVPECPEnpp1 FERTFSN...CRCDAACVSLGNCCLDFQETCVEPTHIWTCNKFRCGEKRLSRFVCSCADDCKTHNDCCINYSSVCQDKKSWVEETCESIDTPECPEnpp3 FDSSHRGLEGCRCDSGCTGRGDCCWDFEDTCVKSTQIWTCNLFRCGENRLETALCSCADDCLQRKDCCADYKTVCQGESPWVTEACASSQEPQCPEnpp4 MFNMKILVIPLFWGLVTGYKGNSEnpp5 MIPEFLLASCTLATLCHSAPFSEnpp6 MAAKLWTFLLGFGLSWVWP...Enpp7 MGHSAVLLCVALAILPACVTGA

α1 α2 η1 α3 α4 η2

Enpp1 DFTQPADKCDGPLSVSSFILPHRPDNDESCNSSEDESKWVEELMKMHTARVRDIEHLTGLDFYRKTSRSYSEILTLKTYLHTYESEI QLSETPLECS.ALESSAYILPHRPDNIESCTHGKRESSWVEELLTLHRARVTDVELITGLSFYQDRQESVSELLRLKTHLPIFSQED DQTHTPDSCPGWLDVLPFIVPHRPTNIESCSENKTEDLWVEERFQAHAARVRDVELLTGLDFYQEKAQPVSQILQLKTYLPTFETII

LSSSP.EAKYDAFLVTNMVPMYPAFKRVWTYFQRVLVKKYASERNGVNVISGPIFDYNYNGLRDIEDEIKQY...VEGSSIPVPTHYYSIITSCLLNRVSNHIYSEALLTSNIVPMYQSFQVIWHYLHDTLLQRYAHERNGINVVSGPVFDFDYDGRYDSLEILKQNSRVIRSQEILIPTHFFIVLTSCKKGTN..ESRYDALITSNLVPMYKEFKKMWDYFHEVLLIKYAIERNGLNVVSGPIFDYNYDGHFDAPDEITQY...VAGTDVPIPTHYFVVLTSCK

TEERHLLYGRPAVLYRTS.YDILYHTDFESGYSEIFLMPLWTSYTISKQAEVSSIPEHLTNCVRPDVRVSPGFSQNCLAYKNDKQMSYGFLFPPYIYHMTVPYGRPRILLKQHRVCLLQQQQFLTGYSLDLLMPLWASYTFLSNDQFS..RDDFSNCLYQDLRIPLSPVHKCSYYKSNSKLSYGFLTPPRTVKANLPFGRPRVMQKNGDHCLLYHRDYISGYGKAMKMPMWSSYTVLKPGDTSSLPPTVPDCLRADVRVAPSESQKCSFYLADKNITHGFLYPAI


Supplementary Figure 1 ATX–LPA signaling pathway and vertebrate Enpp family. (a) Schematic illustration of the ATX–LPA signaling pathway. Note that the acyl chains of the LPC substrates vary in length and saturation. (b) Domain organization and substrates of vertebrate Enpp family members. The cell membrane is shown as a two gray lines. ATX (Enpp2) is a secreted lysoPLD, which consists of an amino-terminal signal sequence, two SMB-like domains, a catalytic domain and a carboxyl-terminal nuclease-like domain. Enpp1 and Enpp3 are type II transmembrane proteins that share a similar domain organization, except for an additional N-terminal intracellular domain and a single transmembrane domain. Enpp4–7 are type I transmembrane or glycosylphosphatidylinositol (GPI)-anchored proteins, consisting of an N-terminal signal sequence, a catalytic domain and a C-terminal hydrophobic domain, but lacking the nuclease-like domain. (c) Multiple sequence alignment of ATX proteins from different vertebrate species. (d) Multiple sequence alignment of mouse Enpp1–7. In c and d, the catalytic Thr residue (Ser for Enpp6) is indicated by a red triangle. Residues involved in Zn2+, Ca2+, Na+ and K+ coordination are indicated by gray, yellow-green, purple and blue triangles, respectively. The essential Cys residues (Cys413 and Cys801) and N-glycosylation sites are indicated by brown and yellow triangles, respectively. Residues involved in LPA recognition and hydrophobic pocket formation are indicated by green triangle and squares, respectively. Residues involved in hydrophobic channel formation are indicated by green circles. The N-terminal signal sequence is indicated by a gray bar. Signal peptidase and proprotein convertase cleavage sites are indicated by gray and red arrows, respectively. The secondary structures are shown above the sequences. Insertion loop sequences are highlighted by the pink background in d.


Supplementary Figure 2 SMB-like domain. (a) Superposition of the two ATX SMB-like domains. (b) Multiple sequence alignment of the ATX SMB-like domains and the vitronectin (VN) SMB domain. Disulfide linkages are indicated by black lines. Residues involved in PAI-1 binding in the VN SMB domain are indicated by red triangles. (c) Superposition of the ATX SMB1 (orange) and the VN SMB domain (PDB ID 1OC0, gray) in complex with PAI-1 (red). (d) Superposition of the ATX SMB2 (brown) and the VN SMB domain (PDB ID 1OC0, gray) in complex with PAI-1 (red).

SMB1 51 C K RC E C C C CC CWTNTSGS . G F LQEVGPPD R DNL KSYSS HDFDEL L...........SMB2 95 C K RC E C C C CC CKTARGWE T D G VRNEEN.A H SED LSRGD TNYQVV KG..........VN-SMB 1 C K RC E C C C CC C...DQES . G T GFNVDK.K Q DEL SYYQS TDYTAE KPQVTRGDVFTM

Ser81Tyr27

Glu23

Arg111Phe13

Asn77

Leu78Leu24

SMB1

SMB2

Asn53-glycan

Core

PAI-1

Cys73Cys117

Asn53

Cys93Cys137

Cys62Cys107

Cys58Cys102

Cys75Cys119

Cys85Cys129

Cys86Cys130

Cys79Cys123

NAG

NAG

51

94

95

139

SMB1

VN SMB

SMB2

VN SMB

Core

PAI-1

Tyr82Tyr28

Ser81Tyr27

Thr10

Phe63

Gln66

Glu23

Phe13

Arg111Phe13

Asn77

Leu78Leu24

Tyr27

Arg126Tyr28

Gly108Thr10

Glu121Glu23

Asp122Leu24

a

b

c

Ser125

d


16:0-LPA

α10

Tyr306

Trp275

Thr209

Asn230Lys208

Asp473

Phe210

Trp254Tyr214

Ala217Leu243

Leu213

Leu216

β7

β6

β1

α6α13

β16

Met512Leu259

Phe27318:1-LPA

α10

Tyr306

Trp275

Thr209

Asn230Lys208

Asp473

Phe210

Trp254Tyr214

Ala217Leu243

Leu213

Leu216

β7

β6

β1

α6α13

β16

Met512Leu259

Phe273

14:0-LPA16:0-LPA18:1-LPA18:3-LPA22:6-LPA

SO42-

D171

Zn2+

T209H474

H315D311

H359

D358

L216

ba

c d

Thr209

His474 SO42-

His315

Asp311

Zn2+

Asp171

His359

Asp358Zn2+

Thr209

His474

His315Asp311

Asp171

His359

Asp358

Glu308

Leu216

Free14:0-LPA16:0-LPA18:1-LPA18:3-LPA 22:6-LPA

16:0-LPA

α10

Tyr306

Trp275

Thr209

Asn230Lys208

Asp473

Phe210

Trp254

Trp260

Tyr214

Ala217Leu243

Leu213

Leu216

Glu308

Asp311

β7

β6

β1

α6α13

β16

Met512Leu259

Ile167

Phe27318:1-LPA

α10

Tyr306

Trp275

Thr209

Asn230Lys208

Asp473

Phe210

Trp254

Trp260

Tyr214

Ala217Leu243

Leu213

Leu216

Glu308

Asp311

β7

β6

β1

α6α13

β16

Met512Leu259

Ile167

Phe273

Supplementary Figure 3 Active site. (a) Anomalous difference density map for the bound Zn2+ ions, contoured at 15σ (magenta). Coordinations to the Zn2+ ions are shown as dashed yellow lines. Diffraction data were collected at a wavelength of 1.2824 Å at 2.2 Å resolution. (b) Superposition of the ATX active sites in the free-form (white) and in complex with 14:0-LPA (gray), 16:0-LPA (orange), 18:1-LPA (blue), 18:3-LPA (magenta) and 22:6-LPA (green). The Zn2+ ions are shown as spheres. (c, d) Structures of ATX in complex with 16:0-LPA (c) and 18:1-LPA (d). The bound LPA molecules are depicted by green stick models. FO – FC omit electron density maps, contoured at 2.5σ, are shown as a yellow mesh. Hydrogen bonds are shown as dashed lines.


Supplementary Figure 4 Catalytic domain. (a) Structure-based sequence alignment of the ATX catalytic domain and X. axonopodis NPP. The Zn2+-coordinating residues are indicated by gray triangles. The catalytic Thr residue and the conserved Asn and Tyr residues are indicated by red and green triangles, respectively. The insertion loop sequence is highlighted in pink. (b) Superposition of the ATX catalytic domain (cyan) and X. axonopodis NPP (PDB ID 2GSN, gray). The insertion loop of X. axonopodis NPP is highlighted in pink. The bound Zn2+ ions are shown as spheres.

170 180 190 200 210 220 230 240

ATX L S DG RA G PN L G A M P YP TFPN YTL TGL P HGIV NSM DP ...VRPP IIF V F SYMKK SKVM IEK RSC TH PY R V TK L A Y ES G Y VFDATFHLRGREXax L S DG RA G PN L G A M P YP TFPN YTL TGL P HGIV NSM DP SASTPHA LLI I L DMLDR ..IT LSH ARE VR RW A S SL H V R DH H R TLGGFWLSKSEA

250 260 270 280 290 300 310

ATX RWWGG P W G A WS R WL R Y E D GH KFNH Q L ITATKQ VR GTFF ...................VS.IPHER ILTILQ SLPDNE PSVYAF S QP FS KYXax RWWGG P W G A WS R WL R Y E D GH VGDA E V VGVENT QH .AT. WPGSEAAIKGVRPSQWRHYQKGVRLDT VDAVRG ATDGAQ NRLVTL F HV EA DH

320 330 340 350 360 370 380 390 410

ATX GP R D G L G N I V DHGM V T FGPEMTNPL EI KTV Q MD LKQLKLHRCV V F G ED TCDRTEFLSNYLTNVDDI LVPGTLGRIRPKIPNNLKYDPKAIXax GP R D G L G N I V DHGM V T ESRQYADAV AV AAI R LA MQRDGTRART I V S AE APGHAISVEDIAP.PQIA AITDGQVIGFEP......LPGQQA

410 420 430 440 450 460 470 480 490

ATX A H K LP R Y RI L W P K G HG D SM VF GP I NLTCKKPDQ FKPYM QH K LH ANNR ED HLLVERR HVARK LDVY KPSGKCFFQ D F NKVN QT VGY TFKYRXax A H K LP R Y RI L W P K G HG D SM VF GP A EASVLGAHD YDCWR AE A WQ GSHP PS VCQMHEG DAL.F DKLA RAQ..RGTR S Y PALP RA LAQ DLAQG

500 510 520 530

ATX P F N Y M LLG APN G L LR TKV P E IEL NV CD LKP N THGS NHL ........ Xax P F N Y M LLG APN G L LR KTL G D VDV AL SR IPA D NPAT LPA MPPAPDAR

β1 η3 α5 β2 β3 α6 α7 β4 β5

η4 η5 α8 α9 β6 α10

η6 α11 β7 β8 β9 η7 η8 β10 β11 α12

β12 η9 η10 β13 β14 η11 β15 η12 β16

β17 β18 η13 α13 η14

β1 η1 α1 β2 β3 α2 α3 β4 β5 α4

η2 α5 β6 α6 η3 β7 α7

α8 η4 β8 β9 β10 α9 β11 β12

α10 β13 β14 η5 η6 β15 β16 α11 β17 η7 β18

β19 β20 η8 α12 η9

α8α5

α9

β6

α6

a

b

Insertion loop

ATX

X. ax. NPP

α8α5

α9

β6

Tyr306Tyr205

Thr209Thr90

Asn230Asn111

His474His363

His315His214 Asp311

Asp210Asp171Asp54

H359H258

Asp358Asp257

α6


Supplementary Figure 5 Nuclease-like domain. (a) Ca2+ binding to the nuclease-like domain. The Ca2+ ion and water molecules are shown as green and red spheres, respectively. The 2FO – FC electron density map, contoured at 2σ, is shown as a gray mesh. (b) K+ binding to the nuclease-like domain. The K+ ion and water molecules are depicted by blue and red spheres, respectively. The 2FO – FC electron density map, contoured at 1σ, is shown as a gray mesh. (c) Na+ binding to the nuclease-like domain. The Na+ ion and water molecules are depicted by purple and red spheres, respectively. The 2FO – FC electron density map, contoured at 2σ, is shown as a gray mesh. (d) Structure-based sequence alignment of the ATX nuclease-like domain and Anabaena NucA. Residues involved in Ca2+, K+ and Na+ binding in ATX are indicated by yellow-green, blue and purple triangles, respectively. (e) Superposition of the ATX nuclease-like domain (magenta) and Anabaena NucA (PDB ID 1ZM8, gray). The Ca2+, K+ and Na+ ions bound to ATX are shown as green, blue and purple spheres, respectively. The residues essential for the nuclease activity of NucA, and the corresponding residues of ATX, are depicted by sticks.

Na+

Ca2+

Wat

Asn737

Asp735

Met671

Leu741

Asp668

Asn739

Asp743

K+

Asn797Ser803

Wat

WatSer800

K+

Na+

Lys705Asn155

Ile640 Arg93

a b

Na+

Ca2+

Wat

Asn737Wat

Asp735

Met671

Leu741

Asp668

Asn739

Asp743

d

c

e

K+

K+

Wat

Tyr665

Wat

Asn797Ser803

Wat

Cys413

Cys801

WatSer800

Na+

NLD

NucA

Lys705Asn155

Ca2+

Ile640 Arg93

Wat

590 600 610 620 630 640 650 660

ATX HLL G P T Y T Q S RPD G ..........STEER Y R AVLYR SYDILYHTDFESG SEIFLMPLW SYTISK AEV SIP.EHLTNCV VRVSP FSQNCLANucA HLL G P T Y T Q S RPD G MQVPPLTELSPSISV L N SGATP KLTPDNYLMVKNQ ALSYNNSKG ANWVAW LNS WLGNAERQDNF KTLPA WVRVTPS

670 680 690 700 710 720 730 740 750

ATX G P E FL TNM P P R S GP YKNDKQMSY FLF .PYLSSSP AKYDA V V MY AFK VWTYFQRVLVKKYA ERNGVNVIS IFDYNYNGLRDIEDEIKQYVENucA G P E FL TNM P P R S GP MYSGSGYAR HIA SADRTKTT DNAAT M M QT DNN NTWGNLEDYCRELV QGKELYIVA N............GSLGKPLK

760 770 780 790 800 810 820 830 840

ATX G VP I P G I P E V E LTG DF S SSIP THYYS ITSCLDFTQ ADKCD PLSVSSF LPHR DND SCNSSEDESKWVEELMKMHTAR RDI H L YRKT RSYSENucA G VP I P G I P E V E LTG DF S .KVT KSTWK VVV...LDS GSGLE ITANTRV AVNI NDP ...........LNNDWRAYKVS DEL S Y LSNV PNIQT

850

ATX K ILTL TYLHTYESEI NucA K SIES VDN.......

α14 β19 β20 β21 β22 η17 η18 α15

β23 η19 η20 η21 β24 α16 α17 β25 η22

β26 β27 η23 α18 β28 α19 β29

α20

η1 β1 β2 β3 η2 η3

β4 η4 α1 η5 β5 α2 α3 β6

β7 η6 β8 η7 α4 α5


Supplementary Figure 6 N-glycosylation. (a–c) 2FO – FC electron density maps for the N-glycans at Asn53 (a), Asn410 (b) and Asn524 (c), shown as gray meshes contoured at 1σ.

MAN

NAG

MAN

MANAsn524

c

BMA

NAG

a

NAG

Asn53

NAG

Asn410

bNAG

NAG


Supplementary Figure 7 Interdomain interaction. (a) Interface between the catalytic and nuclease-like domains. The Ca2+, Na+ and well-ordered water molecules are shown as green, purple and red spheres. The N-glycans at Asn410 and Asn524 are shown as stick models. The essential interdomain disulfide linkage between Cys413 and Cys801 is depicted by a stick model. (b) The well-ordered water molecules at the domain interface. Water molecules are shown as red spheres. The 2FO – FC electron density map, contoured at 1.5σ, is shown as a gray mesh. (c) Interaction between the L1 linker and the catalytic domain. (d) Interaction between the L2 linker and the nuclease-like and catalytic domains. Hydrogen bonds are shown as dashed lines in panels c and d.

L2

Ins

NLDL2

Asn410-glycan

Asn524-glycan

Cys413

Cys801

Ins

Core

NLD

a

Ins

Core

NLD

Ins

Core

Leu845

Thr526

Asp824

Glu507

Arg821

Thr849

NLD

Glu504

Tyr221

Ser529

Ile506

His433

b

c

L2

Ca2+

Core

NLD

L1L1

Core

SMB2

d

L2

Ca2+

Pro544

Met556

Cys662

Cys566

Val547

Ser548

Glu546Tyr601

Tyr605

Asn537

Thr603

Val599

Val652

His643

Cys647Cys568

Tyr689

Arg649

Asp606 Asn551

L1

Core

Lys343

Met339

Tyr490

Arg299

Gly159Cys350

Cys148

Asp149

α11

Glu155

Glu140

α9

Asp146

Lys190

Asp340

Val347

Val351 Leu191Pro157

Arg349

L2

Ins

NLD

Na+

L2

Ca2+

Asn410-glycan

Asn524-glycan

Cys413

Cys801

Ins

Core

NLD

a

Ins

Core

NLD

Ins

Core

Leu845

Thr526

Asp824

Glu507

Arg821

Thr849

NLD

Glu504

Tyr221

Ser529

Ile506

His433

b

c

L2

Ca2+

Core

NLD

L1L1

Core

SMB2

d

Asn524-glycan

L2

Ca2+

Core

Asp514His527

Phe539

Asn531

Thr567

Pro544

Thr536

Thr587

Val648 Met556

Tyr557

Cys662

Cys566

Val547

Arg835

Ile607

Ser548

Leu564

Glu546

Asn572

Tyr601

Tyr605

Asn537

Lys584

Thr603

Val599

Arg596

Glu686

NLD

His582Asp690

Asp570

Val652

Glu575

Leu663

His643

Cys647Cys568

Tyr689

Arg649

Asp606 Asn551

L1L1

Core

Lys343

Arg535

Met339

Trp143

Tyr490

Leu292Arg299

Gly159

Cys156

Cys350

Cys194

Cys148

Glu150

Asp149

Ser141α11

Phe160

Glu155

Glu140

α9

SMB2

Asp146

Lys190

Asp340

Leu347

Val351 Leu191

Pro164

Pro157

Arg349


0

10

20

30

40

50

Lys

oPLD

act

ivity

(nm

ol µ

g–1 h

–1)

MouseHuman

12:0

14:0

16:0

18:0

18:1

18:2

18:3

LPC substrates

LPA

prod

uced

(µM

)

a b c

dLy

soPL

D a

ctiv

ity (n

mol

µg–1

h–1

)

pH pH

5 6 7 8 9 100

50

100

150

200

0

5

10

15

5 6 7 8 9 10

14:0

16:0

18:0

18:1

18:2

18:3

20:4

22:6

14:012:0

16:0

18:0

18:1

18:2

18:3

0

0.05

0.1

0.15

0.2

0.25

0

0.05

0.1

0.15

0.2

0.25

Wild typeLI1LI2LI3

Wild typeF210AL213AN230AL243AF249AW254AF274AY306AM512A0

0.05

0.1

0.15

0 100 200 300 400 500 0 100 200 300 400 500

0 100 200 300 400 5000 100 200 300 400 500

ATX (ng ml–1) ATX (ng ml–1)

Cel

l mot

ility

(OD

590

nm

)C

ell m

otilit

y (O

D 5

90 n

m)

0

0.05

0.1

0.15

0.2

0.25

MDA-MB-231PC-3

e

400 410 420 430 440 450 460 470 480 490m/z

0

25

50

75

100

Rel

ativ

e ab

unda

nce

18:1

20:4

20:3

22:6

17:0(internal standard)

Supplementary Figure 8 Biochemical and cell biological characterization. (a) LPC specificities of mouse and human ATXs. LPC-hydrolyzing activities were measured in the presence of 0.05% Triton X-100. (b) LPC-hydrolyzing activity of mouse ATX at different pH values, in the presence of 0.05% Triton X-100. Recombinant mouse ATX was incubated with various LPC species at the indicated pH values. (c) LPC-hydrolyzing activity of mouse ATX at different pH values in plasma. Various buffers at the indicated pH values were added to the mouse plasma, and then LPA production was evaluated using mass spectrometry. In a–c, error bars represent s.d. (n = 3). (d) Cell motility-stimulating activity of mouse ATX. The cell motility-stimulating activities of wild-type and each mutant ATX were examined for human prostate cancer cells (PC-3) and human breast cancer cells (MDA-MB-231), using a Boyden-chamber assay, in which the cells were placed in the upper chambers and the wild-type and mutant ATX proteins were in the lower chambers. (e) Mass spectrometric analysis of the purified ATX protein used for crystallization, showing the presence of different LPA molecules (18:1, 20:4, 20:3 and 22:6) endogenously bound to the protein.

F210A

L213A

N230A

L243A

F249A

W254A

F274A

Y306A

M512A

LI1

LI2

LI3

Wild type

PC-3 MDA-MB-231

++

++

–

++

++

++

++

+

++

+

+

+

++

++

++

–

++

++

++

++

+

++

+

+

+

++


supplementary information crystal structure of …...supplementary information crystal structure of...

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