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).
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988
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
Residue Atom Residue Atom Distance (Å)
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
Residue Atom Residue Atom Distance (Å)
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
Residue Atom Residue Atom Distance (Å)
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
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988
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
Residue Atom Residue Atom Distance (Å)
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
Residue Atom Residue Atom Distance (Å)
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
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988
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
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988
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
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988
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
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988
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.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988
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
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988
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.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988
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
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988
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
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988
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
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988
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
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988
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
++
++
–
++
++
++
++
+
++
+
+
+
++
++
++
–
++
++
++
++
+
++
+
+
+
++
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1988