crystal structure of enpp1, an extracellular glycoprotein ... · crystal structure of enpp1, an...

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Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling Kazuki Kato a,1 , Hiroshi Nishimasu a,1 , Shinichi Okudaira b , Emiko Mihara c , Ryuichiro Ishitani a , Junichi Takagi c,2 , Junken Aoki b,2 , and Osamu Nureki a,2 a Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan; b Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan; and c Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan Edited by Paul Schimmel, The Skaggs Institute for Chemical Biology, La Jolla, CA, and approved September 6, 2012 (received for review May 12, 2012) Enpp1 is a membrane-bound glycoprotein that regulates bone mineralization by hydrolyzing extracellular nucleotide triphosphates to produce pyrophosphate. Enpp1 dysfunction causes human dis- eases characterized by ectopic calcication. Enpp1 also inhibits insulin signaling, and an Enpp1 polymorphism is associated with insulin resistance. However, the precise mechanism by which Enpp1 func- tions in these cellular processes remains elusive. Here, we report the crystal structures of the extracellular region of mouse Enpp1 in complex with four different nucleotide monophosphates, at resolu- tions of 2.73.2 Å. The nucleotides are accommodated in a pocket formed by an insertion loop in the catalytic domain, explaining the preference of Enpp1 for an ATP substrate. Structural mapping of dis- ease-associated mutations indicated the functional importance of the interdomain interactions. A structural comparison of Enpp1 with Enpp2, a lysophospholipase D, revealed marked differences in the domain arrangements and active-site architectures. Notably, the Enpp1 mutant lacking the insertion loop lost the nucleotide-hydrolyz- ing activity but instead gained the lysophospholipid-hydrolyzing ac- tivity of Enpp2. Our ndings provide structural insights into how the Enpp family proteins evolved to exert their diverse cellular functions. molecular evolution | X-ray crystallography E npp1 (also known as PC-1) is a type II transmembrane gly- coprotein involved in the regulation of bone mineralization (1, 2). Enpp1 is expressed on the outer surfaces of mineralizing cells, such as osteoblasts and chondrocytes, and on the membranes of osteoblast- and chondrocyte-derived matrix vesicles. Physiological mineralization is regulated by the balance between the extracel- lular concentrations of inorganic phosphate (Pi), a substrate for mineralization, and inorganic pyrophosphate (PPi), an inhibitor of mineralization (3). Enpp1 negatively regulates bone mineraliza- tion by hydrolyzing extracellular nucleotide triphosphates (NTPs) to produce PPi, whereas tissue-nonspecic alkaline phosphatase positively regulates mineralization by hydrolyzing NTPs and PPi to produce Pi. The spontaneous ttw (tiptoe walking) mutant mouse, with a nonsense mutation in the Enpp1 gene, exhibits ectopic os- sication of the spinal ligaments, a phenotype similar to ossica- tion of the posterior longitudinal ligament, which is a common form of human myelopathy caused by ectopic ossication of spinal ligaments (4). Moreover, mutations in the Enpp1 gene are asso- ciated with generalized arterial calcication of infancy (GACI), a severe autosomal-recessive human disorder characterized by calcication of the internal elastic lamina of large- and medium- sized arteries and stenosis (57). Enpp1 reportedly inhibits insulin signaling (817), although controversy remains (1821). Enpp1 is overexpressed in bro- blastic cells from insulin-resistant individuals (8), and Enpp1 overexpression impaired insulin signaling in cultured cells and mice (12, 13). Enpp1 binds directly to the insulin receptor, thereby inhibiting its insulin-induced conformational changes (14). Moreover, the K173Q polymorphism of Enpp1 (often described as K121Q,assuming the use of the ATG start codon 156 bp downstream from the correct one) is associated with insulin re- sistance, type 2 diabetes, and obesity (15, 16). Enpp1 is implicated in a variety of physiological and pathological conditions. However, the precise mechanisms by which Enpp1 participates in these cellular processes remain unclaried because of the lack of structural information. Although Enpp1 is essential for the regulation of physiological mineralization, its substrate specicity for different nucleotides and the molecular mechanism conferring its specicity remain unknown. It also is unclear why mutations of amino acid residues located outside the active site render the enzyme inactive and are associated with GACI. More- over, the molecular mechanism by which Enpp1 inhibits insulin signaling has not been elucidated. Enpp1 is a member of the ectonucleotide pyrophosphatase/ phosphodiesterase (Enpp) family of proteins, which are conserved in vertebrates and hydrolyze pyrophosphate or phosphodiester bonds in various extracellular compounds, such as nucleotides and lysophospholipids (22, 23). The seven mammalian Enpp proteins, Enpp17, have distinct substrate specicities and tissue distri- butions and thus participate in different biological processes. Enpp2 (also known as autotaxin) is a secreted lysophospholipase D (lysoPLD) that hydrolyzes lysophosphatidylcholine (LPC) to produce lysophosphatidic acid (LPA), which in turn activates G protein-coupled receptors to evoke various cellular responses (24). The other Enpp family members are either membrane-bound or glycosylphosphatidylinositol-anchored proteins. Enpp13 are composed of two N-terminal somatomedin B (SMB)-like domains (SMB1 and SMB2), a catalytic domain, and a nuclease-like domain, whereas Enpp47 consist of a catalytic domain and lack the SMB- like and nuclease-like domains. The crystal structures of Enpp2 revealed that lipid substrates are accommodated within a hydro- phobic pocket in the catalytic domain (25, 26), which is occluded by an insertion loop in a bacterial nucleotide pyrophosphatase/phos- phodiesterase from Xanthomonas axonopodis (XaNPP). The Enpp family members (except for Enpp2) also have the corresponding insertion sequence. These observations explained why Enpp2 is the only family member that exhibits lysoPLD activity and suggested Author contributions: H.N., R.I., J.T., J.A., and O.N. designed research; K.K., S.O., and E.M. performed research; K.K., H.N., S.O., R.I., J.A., and O.N. analyzed data; and K.K., H.N., R.I., J.T., J.A., and O.N. wrote the paper. The authors declare no conict of interest. This article is a PNAS Direct Submission. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 4GTW (AMP complex), 4GTX (TMP com- plex), 4GTY (GMP complex), and 4GTZ (CMP complex)]. 1 K.K. and H.N. contributed equally to this work. 2 To whom correspondence may be addressed. E-mail: [email protected], [email protected], or [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1208017109/-/DCSupplemental. 1687616881 | PNAS | October 16, 2012 | vol. 109 | no. 42 www.pnas.org/cgi/doi/10.1073/pnas.1208017109 Downloaded by guest on June 13, 2020

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Page 1: Crystal structure of Enpp1, an extracellular glycoprotein ... · Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling Kazuki

Crystal structure of Enpp1, an extracellularglycoprotein involved in bone mineralizationand insulin signalingKazuki Katoa,1, Hiroshi Nishimasua,1, Shinichi Okudairab, Emiko Miharac, Ryuichiro Ishitania, Junichi Takagic,2,Junken Aokib,2, and Osamu Nurekia,2

aDepartment of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan; bGraduate Schoolof Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan; and cInstitute for Protein Research, Osaka University, Suita,Osaka 565-0871, Japan

Edited by Paul Schimmel, The Skaggs Institute for Chemical Biology, La Jolla, CA, and approved September 6, 2012 (received for review May 12, 2012)

Enpp1 is a membrane-bound glycoprotein that regulates bonemineralization by hydrolyzing extracellular nucleotide triphosphatesto produce pyrophosphate. Enpp1 dysfunction causes human dis-eases characterizedbyectopic calcification. Enpp1 also inhibits insulinsignaling, and an Enpp1 polymorphism is associated with insulinresistance. However, the precise mechanism by which Enpp1 func-tions in these cellular processes remains elusive. Here, we report thecrystal structures of the extracellular region of mouse Enpp1 incomplex with four different nucleotide monophosphates, at resolu-tions of 2.7–3.2 Å. The nucleotides are accommodated in a pocketformed by an insertion loop in the catalytic domain, explaining thepreference of Enpp1 for an ATP substrate. Structural mapping of dis-ease-associatedmutations indicated the functional importance of theinterdomain interactions. A structural comparison of Enpp1 withEnpp2, a lysophospholipase D, revealed marked differences in thedomain arrangements and active-site architectures. Notably, theEnpp1mutant lacking the insertion loop lost the nucleotide-hydrolyz-ing activity but instead gained the lysophospholipid-hydrolyzing ac-tivity of Enpp2. Our findings provide structural insights into how theEnpp family proteins evolved to exert their diverse cellular functions.

molecular evolution | X-ray crystallography

Enpp1 (also known as “PC-1”) is a type II transmembrane gly-coprotein involved in the regulation of bone mineralization (1,

2). Enpp1 is expressed on the outer surfaces of mineralizing cells,such as osteoblasts and chondrocytes, and on the membranes ofosteoblast- and chondrocyte-derived matrix vesicles. Physiologicalmineralization is regulated by the balance between the extracel-lular concentrations of inorganic phosphate (Pi), a substrate formineralization, and inorganic pyrophosphate (PPi), an inhibitor ofmineralization (3). Enpp1 negatively regulates bone mineraliza-tion by hydrolyzing extracellular nucleotide triphosphates (NTPs)to produce PPi, whereas tissue-nonspecific alkaline phosphatasepositively regulates mineralization by hydrolyzing NTPs and PPi toproduce Pi. The spontaneous ttw (tiptoe walking) mutant mouse,with a nonsense mutation in the Enpp1 gene, exhibits ectopic os-sification of the spinal ligaments, a phenotype similar to ossifica-tion of the posterior longitudinal ligament, which is a commonform of humanmyelopathy caused by ectopic ossification of spinalligaments (4). Moreover, mutations in the Enpp1 gene are asso-ciated with generalized arterial calcification of infancy (GACI),a severe autosomal-recessive human disorder characterized bycalcification of the internal elastic lamina of large- and medium-sized arteries and stenosis (5–7).Enpp1 reportedly inhibits insulin signaling (8–17), although

controversy remains (18–21). Enpp1 is overexpressed in fibro-blastic cells from insulin-resistant individuals (8), and Enpp1overexpression impaired insulin signaling in cultured cells andmice (12, 13). Enpp1 binds directly to the insulin receptor, therebyinhibiting its insulin-induced conformational changes (14).Moreover, the K173Q polymorphism of Enpp1 (often described

as “K121Q,” assuming the use of the ATG start codon 156 bpdownstream from the correct one) is associated with insulin re-sistance, type 2 diabetes, and obesity (15, 16).Enpp1 is implicated in a variety of physiological and pathological

conditions. However, the precise mechanisms by which Enpp1participates in these cellular processes remain unclarified becauseof the lack of structural information. Although Enpp1 is essentialfor the regulation of physiological mineralization, its substratespecificity for different nucleotides and the molecular mechanismconferring its specificity remain unknown. It also is unclear whymutations of amino acid residues located outside the active siterender the enzyme inactive and are associated with GACI. More-over, the molecular mechanism by which Enpp1 inhibits insulinsignaling has not been elucidated.Enpp1 is a member of the ectonucleotide pyrophosphatase/

phosphodiesterase (Enpp) family of proteins, which are conservedin vertebrates and hydrolyze pyrophosphate or phosphodiesterbonds in various extracellular compounds, such as nucleotides andlysophospholipids (22, 23). The seven mammalian Enpp proteins,Enpp1–7, have distinct substrate specificities and tissue distri-butions and thus participate in different biological processes.Enpp2 (also known as “autotaxin”) is a secreted lysophospholipaseD (lysoPLD) that hydrolyzes lysophosphatidylcholine (LPC) toproduce lysophosphatidic acid (LPA), which in turn activates Gprotein-coupled receptors to evoke various cellular responses (24).The other Enpp family members are either membrane-boundor glycosylphosphatidylinositol-anchored proteins. Enpp1–3 arecomposed of two N-terminal somatomedin B (SMB)-like domains(SMB1 and SMB2), a catalytic domain, and a nuclease-like domain,whereas Enpp4–7 consist of a catalytic domain and lack the SMB-like and nuclease-like domains. The crystal structures of Enpp2revealed that lipid substrates are accommodated within a hydro-phobic pocket in the catalytic domain (25, 26), which is occluded byan insertion loop in a bacterial nucleotide pyrophosphatase/phos-phodiesterase from Xanthomonas axonopodis (XaNPP). The Enppfamily members (except for Enpp2) also have the correspondinginsertion sequence. These observations explained why Enpp2 is theonly family member that exhibits lysoPLD activity and suggested

Author contributions: H.N., R.I., J.T., J.A., and O.N. designed research; K.K., S.O., and E.M.performed research; K.K., H.N., S.O., R.I., J.A., and O.N. analyzed data; and K.K., H.N., R.I.,J.T., J.A., and O.N. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org [PDB ID codes 4GTW (AMP complex), 4GTX (TMP com-plex), 4GTY (GMP complex), and 4GTZ (CMP complex)].1K.K. and H.N. contributed equally to this work.2To whom correspondence may be addressed. E-mail: [email protected],[email protected], or [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1208017109/-/DCSupplemental.

16876–16881 | PNAS | October 16, 2012 | vol. 109 | no. 42 www.pnas.org/cgi/doi/10.1073/pnas.1208017109

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that the insertion loop contributes to defining the substrate spe-cificities (27).Here, we present the crystal structures of the extracellular

region of mouse Enpp1 in complex with four different nucleotidemonophosphates (NMPs), which explain the observed prefer-ence of Enpp1 for the ATP substrate. Unlike Enpp2, the SMB-like domains are disordered and do not interact with the catalyticdomain in Enpp1, suggesting that the SMB-like domains inEnpp1 and Enpp2 have distinct roles. Structural mapping ofdisease-associated mutations indicated the functional signifi-cance of the interaction between the catalytic and nuclease-likedomains in both Enpp1 and Enpp2.

ResultsSubstrate Specificity. The extracellular region (residues 92–905) ofmouse Enpp1 was overexpressed in HEK293S GnT1− cells asa secreted protein and was purified by P20.1 antibody affinity andgel filtration chromatographies. When ATP was incubated withthe purified protein, the production of AMP and PPi, but notADP and Pi, was detected by mass spectrometry (Fig. 1A), in-dicating that Enpp1 hydrolyzes the phosphodiester bond betweenthe α- and β-phosphate groups of ATP. A kinetic analysis showedthat Enpp1 preferably hydrolyzes ATP (kcat = 16 s−1, Km = 46μM), compared with UTP (kcat = 200 s−1, Km = 4.3 mM), GTP(kcat = 820 s−1, Km = 4.2 mM) and CTP (kcat = 8.7 s−1, Km = 1.2mM) (Fig. S1). We also examined the substrate specificity bymeasuring the p-nitrophenyl thymidine 5′-monophosphate (pNP-TMP)–hydrolyzing activity in the presence of different NMPs.AMP inhibited the pNP-TMP–hydrolyzing activity more potentlythan TMP, GMP, and CMP (Fig. 1B). These results showed thatEnpp1 preferably hydrolyzes ATP to produce AMP and PPi andconfirmed that Enpp1 negatively regulates bone mineralization byhydrolyzing ATP, an abundant extracellular nucleotide.

Overall Architecture. We solved the crystal structures of the ex-tracellular domain of Enpp1 in complex with four different NMPs(AMP, TMP, GMP, and CMP) at resolutions of 2.7–3.2 Å (TableS1). Because these four crystal structures are essentially identical(rmsd values less than 0.2 Å for aligned Cα atoms), we describethe AMP complex structure, unless otherwise stated. The struc-ture consists of a catalytic domain (residues 190–578), a nuclease-like domain (residues 629–902), and two linker regions, L1 andL2 (residues 170–189 and 579–628, respectively) (Fig. 2A and B).The catalytic domain interacts with the nuclease-like domain, andthe L2 linker connects the two domains (Fig. 2B). The structurerevealed that Enpp1 is N-glycosylated at Asn267, Asn323, andAsn567 and that the domain interaction is reinforced by theAsn567-linked glycan and the Cys462–Cys846 disulfide linkage,which correspond to the Asn524-linked glycan and the Cys413–Cys801 disulfide linkage, respectively, in mouse Enpp2 (Fig. 2 Band C) (25, 26). The spatial arrangement of the catalytic andnuclease-like domains is conserved in Enpp1 and Enpp2,

suggesting that the interdomain interactions play similar roles inEnpp1 and Enpp2.

Catalytic Domain. The catalytic domain of Enpp1 is structurallysimilar to those of Enpp2 (25, 26) (PDB ID 3NKM, 48% sequenceidentity, rmsd= 1.1Å for 341Cα atoms) andXaNPP (28) (PDB ID2GSU, rmsd= 1.6Å for 339Cα atoms) (Fig. 2B andC andFig. S2).As in Enpp2 and XaNPP, two zinc ions are bound within the activesite of Enpp1. One zinc ion is coordinated by Asp358, His362, andHis517, and the other is coordinated by Asp200, Thr238, Asp405,and His406 (Fig. 3). A previous mutational analysis confirmed thefunctional significance of these zinc-coordinating residues (29).The α-phosphate group of AMP is bound between the two zincions, consistent with our functional data showing that Enpp1hydrolyzes ATP to produce AMP and PPi (Fig. 1A).

Nuclease-Like Domain. The nuclease-like domain of Enpp1 isstructurally similar to that of Enpp2 (25, 26) (PDB ID3NKM, 42%sequence identity, rmsd = 1.4 Å for 248 Cα atoms) (Fig. 2 B and Cand Fig. S3). As in Enpp2, a calcium ion is coordinated by the sidechains of Asp780, Asp782, Asp784, and Asp788 and the main-chain carbonyl group of Arg786, forming an EF hand-like motif. InEnpp2, Asp735 (corresponding toAsp780 in Enpp1) interacts withLys430 (corresponding to Lys479 in Enpp1), and the K430A mu-tation impaired the protein stability (26). In Enpp1, Asp780interacts with Lys479 in the catalytic domain, suggesting the im-portance of the EF hand-like motif for the interdomain inter-actions in both Enpp1 and Enpp2.

SMB-Like Domains. Unexpectedly, electron densities were not ob-served for the twoSMB-likedomains, and there is sufficient roomtoaccommodate the two SMB-like domains in the crystal lattice. AnSDS/PAGE analysis of the dissolved crystals revealed a single band(∼100 kDa) similar to the purified protein (Fig. S4A), indicatingthat the crystallized proteins contain the SMB-like domains. Thereported Enpp1 SMB1 domain (PDB ID 2YS0) is structurallysimilar to the Enpp2 SMB1 domain (25, 26) (PDB ID 3NKM, 53%sequence identity, rmsd = 1.2 Å for 34 Cα atoms) (Fig. S4B). TheSMB2 domains of Enpp1 and Enpp2 share 48% sequence identity,indicating that the Enpp1 SMB2 domain also adopts a rigid struc-ture. A structural comparison of Enpp1 with Enpp2 indicated thatthe SMB1 domain of Enpp1 cannot interact with the catalytic do-main in theway observed inEnpp2 because of steric clasheswith theinsertion loop (Fig. S4C). In addition, Arg283, Gln290, andGln344in the catalytic domain of Enpp2, which participate in the in-teraction with the SMB2 domain (25), are replaced with Glu330,Glu337, and Asp391, respectively, in Enpp1 (Fig. S4C). Theseobservations suggested that, unlike Enpp2, the spatial arrangementof the SMB-like domains of Enpp1 is not fixed by the interactionwith the catalytic domain. To confirm this idea, we prepared anEnpp1mutant bearing the Turbo3C protease recognition sequencebetween SMB2 (Lys169) and L1 (Lys170), incubated the purifiedmutant protein with the protease, and then performed a pulldown

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Fig. 1. Biochemical characterization. (A) Enpp1 hydrolyzes ATP to produce AMP and PPi. Purified Enpp1 was incubated with ATP, and then the reactionproducts were quantified by mass spectrometry. (B) Inhibition of Enpp1 activity by NMPs. The enzymatic activity was measured in the absence or presence of0.5 mM NMPs, using 4 mM pNP-TMP as a substrate. (C) ATP– and pNP-TMP–hydrolyzing activities of Enpp1 mutants. (D) LPC– and pNP-TMP–hydrolyzingactivities of the wild type and ΔIL mutant of Enpp1. Data are shown as mean ± SD (n = 3).

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assay using P20.1-Sepharose.We found that the SMB-like domainswere not pulled down together with the rest of the protein, sug-gesting that the SMB-like domains do not interact with the catalyticdomain (Fig. S4D). In contrast, the equivalent Enpp2 mutant withthe protease recognition sequence between SMB2 (Glu140) and L1(Ser141) was not expressed in HEK293T cells, suggesting differ-ences in the flexibility between SMB2 and L1 in Enpp1 and Enpp2.Notably, the protease treatment had almost no effect on the enzy-matic activity (Fig. S4E), indicating that the SMB-like domains aredispensable for the enzymatic activity ofEnpp1.Given thatEnpp1 isa type II transmembrane protein, the mobile SMB-like domains ofEnpp1 may act as a molecular anchor that connects the trans-membrane region and the catalytic domain.

Nucleotide Recognition. The crystal structures in complex with thefour different NMPs revealed that the phosphate groups and ri-bose moieties are recognized by the protein in a similar manner,and the nucleobase moieties are sandwiched between the sidechains of Phe239 and Tyr322 (Fig. 3). The F239A and Y322Amutants showed reduced hydrolytic activities for ATP and pNP-TMP (Fig. 1C). The AMP N6 atom is recognized by Trp304 andAsp308 through a water-mediated hydrogen bond network (Fig.

3A) in the AMP complex, and the D308A mutant exhibited de-creased ATP–hydrolyzing activity (Fig. 1C). In contrast, thenucleobase moieties of TMP, GMP, and CMP are not recognizedby Enpp1 through hydrogen-bonding interactions (Fig. 3 B–D).These observations can explain the preference of Enpp1 for ATPas a substrate, as described above (Fig. 1A). Asp308 of Enpp1 isreplaced with Glu160 in XaNPP (Fig. S2C) (28), suggesting dif-ferences in the substrate preferences of Enpp1 and XaNPP (al-though the substrate preference of XaNPP remains unclear).

MolecularDeterminantsof Substrate Specificity.The present structurerevealed that the insertion loop (residues 304–323) participates inthe formation of the substrate-binding pocket, with Trp304 in theWPG motif forming a hydrophobic core with Leu196, Ser198,His242, Ile245, Val246, Trp289, and Thr351 (Fig. 4 A and B). Themain-chain amide groups of Trp304 and Tyr322 hydrogen bondwith the side chains of Ser307 and Asp308, respectively. His242 inEnpp1 corresponds to Leu213 in Enpp2, which participates in theformation of the lipid-binding hydrophobic pocket (Fig. 4 C andD). As previously reported (30), the H242L mutant showed re-duced phosphodiesterase activity (Fig. 1C), indicating the im-portance of the interaction between His242 and Trp304 in the

Catalytic domainNuclease-like domain

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Fig. 2. Overall architecture. (A) Domain organization of mouse Enpp1. (B) Crystal structure of the extracellular domain of Enpp1 in complex with AMP.Catalytic domain, cyan; nuclease-like domain, magenta; L1, wheat; L2, yellow-green; EF hand-like motif, pink; insertion loop, gold. AMP and N-glycans areshown as green and yellow sticks, respectively. The bound zinc and calcium ions are shown as gray and yellow-green spheres, respectively. Disulfide linkagesare shown as sticks. The two SMB-like domains, which were disordered in the crystal structure, are indicated by circles. (C) Crystal structure of Enpp2 incomplex with 14:0-LPA (PDB ID 3NKN); color code as in B. The SMB1 and SMB2 domains are colored orange and brown, respectively.

Tyr322

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Fig. 3. Nucleotide recognition. Active site of Enpp1 in complex with AMP (A), TMP (B), GMP (C), and CMP (D). The bound NMPs are shown as green sticks. FO–FComit electron density maps, contoured at 3.5 σ, are shown as blue meshes. The bound zinc ions and water molecules are shown as gray and red spheres,respectively. Hydrogen bonds and coordinate bonds are shown as dashed gray and yellow lines, respectively.

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formation of the nucleotide-binding pocket. To further examinewhether the insertion loop is the major determinant of the sub-strate specificity, we prepared the Enpp1 mutant lacking residues304–323 (ΔIL mutant) and measured its pNP-TMP– and LPC–hydrolyzing activities. The ΔIL mutant showed drastically re-duced pNP-TMP–hydrolyzing activity compared with the wildtype (Fig. 1D), indicating the importance of the insertion loopfor the nucleotide recognition. Notably, the ΔIL mutant dis-played lysoPLD activity (Fig. 1D), suggesting that a lipid-bind-ing pocket is generated by the deletion of the insertion loop.However, the lysoPLD activity of the ΔIL mutant (0.025 nmolμg−1 h−1) was much lower than that of Enpp2 (38 nmol μg−1 h−1)(25). In Enpp2, the hydrophobic pocket is formed by conservedhydrophobic residues, such as Ile167, Leu213, Leu216, Ala217,Leu259, Trp260, Phe273, Val302, Ala304, and Met512 (25),which correspond to Leu196, His242, Ile245, Val246, Ile288,Trp289, Tyr302, Phe349, Thr351, and Met555, respectively, inEnpp1 (Fig. 4 B and D). Thus, the replacements of His242,Val246, Phe349, and Thr351 in Enpp1 with smaller hydrophobicresidues may be required for the formation of a hydrophobicpocket optimized for accommodating lipid substrates. In addition,Tyr302 of Enpp1 is located at a different position from the cor-responding Phe273 of Enpp2, and Tyr302 interacts with Phe303,Arg331, Ala334, and Trp338 (Fig. 4B). Taken together, theseobservations suggested that, in addition to the loop deletion,amino acid replacements may have been necessary for the mo-lecular evolution of Enpp1 to Enpp2.

DiscussionAlthough Enpp1 hydrolyzes various nucleotide substrates in vitro(31), its substrate preference was unknown. Our functional analysisrevealed that Enpp1 preferentially hydrolyzes ATP to produceAMP and PPi. Moreover, the present structures provide a molec-ular basis for PPi production by Enpp1, through ATP hydrolysis. InEnpp2, lipid substrates are accommodated in a deep, hydrophobicpocket (25, 26). In contrast, in Enpp1, the nucleotide substrates areaccommodated in the pocket formed by the insertion sequence,which occludes the hydrophobic pocket. These structural differ-ences clearly explain why Enpp1 hydrolyzes nucleotides but notlipids. Moreover, the deletion of the insertion sequence of Enpp1resulted in the generationof lysoPLDactivity (albeit lower than thatof Enpp2). A structural comparison of Enpp1 andEnpp2 suggestedthat Enpp2 gained the hydrophobic pocket during the course ofevolution through the deletion of the insertion loop, followed byamino acid replacements. These observations reinforced our pre-vious proposal that the insertion sequence participates in definingthe substrate specificity of the Enpp family proteins.A number of genetic mutations in Enpp1 are associated with

GACI, a human disorder with a hypermineralization phenotype(5–7). Most of these mutations mapped to the catalytic and nu-clease-like domains (Fig. 5 A and B). Among these, the R456Q,L579F, L611V, C726R, N792S, E893X, and Y901S mutationsabolished the enzymatic activity in human Enpp1 (5, 32). Thestructure of mouse Enpp1 revealed that most of these residuesparticipate in intradomain or interdomain interactions (Fig. 5 B–G). In the catalytic domain, Arg438 (Arg456; equivalent residues

Insertion loopInsertion loop

Trp304Trp304

Asn323Asn323

Tyr322Tyr322

Pro305Pro305

LPALPA

AMPAMP

Hydrophobic pocketHydrophobic pocketSMB1SMB1

Insertion loop

Trp304

Asn323

Tyr322

Asn323

Tyr322

Pro305

LPA

AMP

AMP

Zn2+

Zn2+

Zn2+

Zn2+Zn2+

Zn2+

Zn2+

Zn2+

Hydrophobic pocketSMB1

Phe239

His242

Ser198 Asp308

Ser307Trp304

Pro305Gly306 Trp289

Phe349

Ile288Val246Ile245

Leu196

Trp338

Ala334

Tyr302

Arg331

Phe303

Thr351

Met555

Phe210 Ala217

Trp260

Leu213

Leu216Leu259Met512

Ser169

Ile167

Ala304

Phe273

Val302

LPA

Phe210 Ala217

Trp260

Leu213

Leu216Leu259Met512

Ser169

Ile167

Ala304

Phe273

Val302

LPA

Asn323

Tyr322

AMP

Phe239

His242

Ser198 Asp308

Ser307Trp304

Pro305Gly306 Trp289

Phe349

Ile288Val246Ile245

Leu196

Trp338

Ala334

Tyr302

Arg331

Phe303

Thr351

Met555

A B

C D

Fig. 4. Substrate binding pocket. (A) Molecular surface of Enpp1. The insertion loop is shown as a tube. (B) Nucleotide-binding pocket of Enpp1 (stereo view).The insertion loop is shown in gold in A and B. (C) Molecular surface of Enpp2 (PDB ID 3NKN). (D) Lipid-binding hydrophobic pocket of Enpp2 (PDB ID 3NKN)(stereo view). The SMB-like domains are omitted for clarity in D. The bound zinc ions are shown as gray spheres in B and D.

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Page 5: Crystal structure of Enpp1, an extracellular glycoprotein ... · Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling Kazuki

of human Enpp1 are indicated in parentheses) hydrogen bondswith Asp258 and Glu490 (Fig. 5C), and Leu561 (Leu579) formsa hydrophobic core with Thr291, Ala292, Gln295, Val297, andLeu559 (Fig. 5D). The D276N mutation in Aps276 (equivalent toAsp258 of mouse Enpp1) also is associated with GACI. In thenuclease-like domain, Glu873 (Glu893) interacts with Lys895 andPhe880 (Fig. 5E). Asn772 (Asn792) andTyr881 (Tyr901) hydrogenbond with Thr676 and with Thr816 and Ser833, respectively (Fig.5E). These observations suggested that these inactivating muta-tions result in either the loss of interactions or steric clashes withinthe individual domains, highlighting the functional significance ofthe catalytic and nuclease-like domains. The ttwmouse carries theG568stop nonsense mutation, which results in the deletion of thenuclease-like domain (4), and the Enpp1 mutant protein witha truncated nuclease-like domain (Δ804–905) is catalytically in-active (18), further highlighting the functional significance of thenuclease-like domain. Leu593 (Leu611) on the L2 linker interactswith Arg641 and Leu653 in the nuclease-like domain, and Arg641interacts with Glu589 on the L2 linker (Fig. 5E). Cys706 (Cys726)in the nuclease-like domain forms a disulfide bond with Cys607 onthe L2 linker (Fig. 5F). These observations indicated that the in-teraction between the nuclease-like domain and the L2 linker isimportant for the enzymatic activity. In addition, disease-causingmutations are mapped at the domain interface (H500P in thecatalytic domain and D804H and R888W in the nuclease-likedomain) (Fig. 5G). His482 (His500) and Arg868 (Arg888) hydro-gen bond with Asp871 and with Tyr250 and Glu547, respectively.Asp784 (Asp804) is located in the EF hand-like motif, and Asp780within this motif interacts with Lys479 in the catalytic domain.These observations indicated the functional significance of theinteraction between the catalytic and nuclease-like domains. Thestructural mapping of the disease-causing mutations revealed thatthe integrity not only of the individual domains but also of theinterdomain interactions is important for the enzymatic activityof Enpp1.Enpp1 interacts directly with the insulin receptor and prevents

insulin-induced conformational changes in the receptor, therebyinhibiting insulin signaling (14). The Enpp1 K173Q polymorphismis associated with obesity and type 2 diabetes (15), and the K173Q

mutant protein inhibited insulin signaling more efficiently, byinteracting with the insulin receptor more strongly than the wildtype (17). Lys173 is replaced by a histidine residue in mouseEnpp1, suggesting that the K173Q polymorphism is specific tohumanEnpp1.Lys173 of humanEnpp1 is locatedwithin the SMB2domain, and thus this domain may participate in the interactionwith the insulin receptor. The SMB2 domain of Enpp2 binds tointegrins (26), and the SMB domain of vitronectin binds to plas-minogen activator inhibitor-1 (PAI-1) (33) and the urokinase re-ceptor (uPAR) (34). Lys173 of human Enpp1 corresponds toArg126 of the mouse Enpp2 SMB2 domain and Tyr28 of thevitronectin SMB domain (Fig. S4B). Arg126 of Enpp2 is exposedto the solvent (25), and Tyr28 of vitronectin participates in theinteraction with PAI-1 (33) and uPAR (34). These observationssupport the notion that Lys173 of human Enpp1 participates in theinteraction with the insulin receptor.We previously hypothesized that the interaction between the

catalytic and nuclease-like domains contributes to maintaining thestructural integrity of the hydrophobic pocket in Enpp2 (25).However, the present results revealed that the interdomain in-teraction is conserved in Enpp1 and is important for the enzymaticactivity, although Enpp1 lacks a hydrophobic pocket. Recent mo-lecular dynamics simulations revealed that the interdomain in-teraction contributes mainly to the correct positioning of thecatalytic threonine residue in the catalytic domain, explaining therequirement of the conserved interdomain interaction in Enpp1and Enpp2 (35). We also found that the SMB-like domains ofEnpp1 do not interact with the catalytic domain (Fig. 2B) and aredispensable for the enzymatic activity (Fig. S4E), consistent withthe mapping of most of the disease-causing mutations on the cat-alytic and nuclease-like domains (Fig. 5A) (5–7). These observa-tions suggested that the SMB-like domains of Enpp1 act asa flexible molecular anchor, consistent with the notion that theSMB2 domain of human Enpp1 interacts with the insulin receptor.In Enpp2, the SMB-like domains interact with the catalytic domainand form a hydrophobic channel, which may serve as an exit forlipid products to specific G protein-coupled receptors (25, 26).Thus, the distinct arrangements of the SMB-like domains are likelyto reflect their functional differences in Enpp1 and Enpp2.

IL

R456Q(Arg438)

L611V(Leu593)

N792S(Asn772)

C726R(Cys706)

1 77 100 142 187 208 597 648 925

L579F(Leu561)

E893X(Glu873)

Y901S(Tyr881)

Cytosolicdomain

SMB1 SMB2TM L1 Catalytic domain L2 Nuclease-like domain

EF hand

Arg438Asp258

Glu490

Leu561Tyr552

Thr291Ala292

Gln295 Val297

Leu559

Asn772

Tyr881

Thr676 Thr816 Ser833

Glu873

Lys895

Lys895Phe880

Phe880

Leu593

Leu653

Glu589

Arg641 Cys706

Cys607

His482

Arg868Glu873

Asp871

Tyr250

Glu547

Asp780

Lys479

Ca2+ Asp784

A B

C

D

GF

E

C D E F G

P305TC126R

Human Enpp1 P250L

Y252Δ

S216YG242F

G266VY261X

D276N

S287F

R481W

Y471CR476X

H500PS504R

Y513C R774CH777R D804H

R821P

Y312XR349K

G342V

Y371F

Y570CY659C

E668K R888WG568stop(in mouse)

Fig. 5. Structural mapping of disease-causing mutations. (A) Mapping of mutations associated with GACI on the primary structure of human Enpp1. (B)Mapping of the disease-causing mutations on the crystal structure of mouse Enpp1. (C–G) Close-up views of boxed areas in B. The residues of mouse Enpp1corresponding to the disease-associated residues of human Enpp1 are shown as gray sticks in B–G.

16880 | www.pnas.org/cgi/doi/10.1073/pnas.1208017109 Kato et al.

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Page 6: Crystal structure of Enpp1, an extracellular glycoprotein ... · Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling Kazuki

In summary, our findings suggest that Enpp1 participates indifferent biological processes through distinct sets of domains: thecatalytic and nuclease-like domains for bone mineralization andthe SMB-like domains for insulin signaling. Moreover, our find-ings indicate that Enpp1 and Enpp2 exert diverse cellular func-tions because of their distinct domain arrangements and active-site architectures, although they share similar primary structures.

Materials and MethodsThe extracellular region (residues 92–905) of mouse Enpp1 was expressed,purified, and crystallized as described previously (36). X-ray diffraction datawere collected at 100 K on beamline BL32XU at SPring-8 (Hyogo, Japan). Thecrystal structure in complex with AMP was determined by the single-wave-length anomalous dispersion method, and the crystal structures in complexwith the other NMPs were determined by molecular replacement. Data

collection and refinement statistics are provided in Table S1. Moleculargraphics were prepared using CueMol (http://www.cuemol.org). Detailedmethods are described in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank the beamline staff at BL32XU at SPring-8for technical help during data collection. This work was supported by grantsfrom the Japan Society for the Promotion of Science (JSPS) through its Fund-ing Program for World-Leading Innovative Research and Development onScience and Technology (FIRST) program (to O.N.); from the Japan Scienceand Technology Agency (JST) through the Core Research for EvolutionalScience and Technology (CREST) program on the Creation of Basic MedicalTechnologies to Clarify and Control the Mechanisms Underlying Chronic In-flammation (to J.A. and O.N.); by a Grant-in-Aid for Scientific Research onInnovative Areas from the Ministry of Education, Culture, Sports, Science andTechnology (MEXT) (to R.I. and O.N.); by a Grant-in-Aid for Young Scientists(A) from MEXT (to H.N.); and by a Grant-in-Aid for Scientific Research (S)from MEXT (to O.N.).

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