Crystal Structure of Enpp1, an Extracellular Glycoprotein Involved in Bone Mineralization and Insulin Signaling

Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling

Kazuki 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 School of 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 bone as “K121Q,” assuming the use of the ATG start codon 156 bp mineralization by hydrolyzing extracellular nucleotide triphosphates downstream from the correct one) is associated with insulin re- to produce pyrophosphate. Enpp1 dysfunction causes human dis- sistance, type 2 diabetes, and obesity (15, 16). eases characterized by ectopic calcification. Enpp1 also inhibits insulin Enpp1 is implicated in a variety of physiological and pathological signaling, and an Enpp1 polymorphism is associated with insulin conditions. However, the precise mechanisms by which Enpp1 resistance. However, the precise mechanism by which Enpp1 func- participates in these cellular processes remain unclarified because tions in these cellular processes remains elusive. Here, we report the of the lack of structural information. Although Enpp1 is essential crystal structures of the extracellular region of mouse Enpp1 in for the regulation of physiological mineralization, its substrate complex with four different nucleotide monophosphates, at resolu- specificity for different nucleotides and the molecular mechanism tions of 2.7–3.2 Å. The nucleotides are accommodated in a pocket conferring its specificity remain unknown. It also is unclear why formed by an insertion loop in the catalytic domain, explaining the mutations of amino acid residues located outside the active site preference of Enpp1 for an ATP substrate. Structural mapping of dis- render the enzyme inactive and are associated with GACI. More- ease-associated mutations indicated the functional importance of the over, the molecular mechanism by which Enpp1 inhibits insulin BIOCHEMISTRY interdomain interactions. A structural comparison of Enpp1 with signaling has not been elucidated. Enpp2, a lysophospholipase D, revealed marked differences in the Enpp1 is a member of the ectonucleotide pyrophosphatase/ domain arrangements and active-site architectures. Notably, the phosphodiesterase (Enpp) family of proteins, which are conserved Enpp1 mutant lacking the insertion loop lost the nucleotide-hydrolyz- in vertebrates and hydrolyze pyrophosphate or phosphodiester ing activity but instead gained the lysophospholipid-hydrolyzing ac- bonds in various extracellular compounds, such as nucleotides and tivity of Enpp2. Our findings provide structural insights into how the lysophospholipids (22, 23). The seven mammalian Enpp proteins, Enpp family proteins evolved to exert their diverse cellular functions. Enpp1–7, have distinct substrate specificities and tissue distri- butions and thus participate in different biological processes. molecular evolution | X-ray crystallography Enpp2 (also known as “autotaxin”) is a secreted lysophospholipase D (lysoPLD) that hydrolyzes lysophosphatidylcholine (LPC) to npp1 (also known as “PC-1”) is a type II transmembrane gly- produce lysophosphatidic acid (LPA), which in turn activates G Ecoprotein involved in the regulation of bone mineralization (1, protein-coupled receptors to evoke various cellular responses (24). 2). Enpp1 is expressed on the outer surfaces of mineralizing cells, The other Enpp family members are either membrane-bound such as osteoblasts and chondrocytes, and on the membranes of or glycosylphosphatidylinositol-anchored proteins. Enpp1–3are osteoblast- and chondrocyte-derived matrix vesicles. Physiological composed of two N-terminal somatomedin B (SMB)-like domains mineralization is regulated by the balance between the extracel- (SMB1 and SMB2), a catalytic domain, and a nuclease-like domain, – lular concentrations of inorganic phosphate (Pi), a substrate for whereas Enpp4 7 consist of a catalytic domain and lack the SMB- mineralization, and inorganic pyrophosphate (PPi), an inhibitor of like and nuclease-like domains. The crystal structures of Enpp2 mineralization (3). Enpp1 negatively regulates bone mineraliza- revealed that lipid substrates are accommodated within a hydro- tion by hydrolyzing extracellular nucleotide triphosphates (NTPs) phobic pocket in the catalytic domain (25, 26), which is occluded by to produce PPi, whereas tissue-nonspecific alkaline phosphatase an insertion loop in a bacterial nucleotide pyrophosphatase/phos- Xanthomonas axonopodis positively regulates mineralization by hydrolyzing NTPs and PPi to phodiesterase from (XaNPP). The Enpp produce Pi. The spontaneous ttw (tiptoe walking) mutant mouse, family members (except for Enpp2) also have the corresponding with a nonsense mutation in the Enpp1 gene, exhibits ectopic os- insertion sequence. These observations explained why Enpp2 is the sification of the spinal ligaments, a phenotype similar to ossifica- only family member that exhibits lysoPLD activity and suggested tion of the posterior longitudinal ligament, which is a common form of human myelopathy caused by ectopic ossification of spinal ligaments (4). Moreover, mutations in the Enpp1 gene are asso- Author contributions: H.N., R.I., J.T., J.A., and O.N. designed research; K.K., S.O., and E.M. fi performed research; K.K., H.N., S.O., R.I., J.A., and O.N. analyzed data; and K.K., H.N., R.I., ciated with generalized arterial calci cation of infancy (GACI), J.T., J.A., and O.N. wrote the paper. a severe autosomal-recessive human disorder characterized by The authors declare no conflict of interest. calcification of the internal elastic lamina of large- and medium- This article is a PNAS Direct Submission. sized arteries and stenosis (5–7). – Data deposition: The atomic coordinates and structure factors have been deposited in the Enpp1 reportedly inhibits insulin signaling (8 17), although Protein Data Bank, www.pdb.org [PDB ID codes 4GTW (AMP complex), 4GTX (TMP com- controversy remains (18–21). Enpp1 is overexpressed in fibro- plex), 4GTY (GMP complex), and 4GTZ (CMP complex)]. blastic cells from insulin-resistant individuals (8), and Enpp1 1K.K. and H.N. contributed equally to this work. overexpression impaired insulin signaling in cultured cells and 2To whom correspondence may be addressed. E-mail: [email protected], mice (12, 13). Enpp1 binds directly to the insulin receptor, thereby [email protected], or [email protected]. inhibiting its insulin-induced conformational changes (14). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Moreover, the K173Q polymorphism of Enpp1 (often described 1073/pnas.1208017109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1208017109 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 that the insertion loop contributes to defining the substrate spe- suggesting that the interdomain interactions play similar roles in cificities (27). Enpp1 and Enpp2. Here, we present the crystal structures of the extracellular region of mouse Enpp1 in complex with four different nucleotide Catalytic Domain. The catalytic domain of Enpp1 is structurally monophosphates (NMPs), which explain the observed prefer- similar to those of Enpp2 (25, 26) (PDB ID 3NKM, 48% sequence ence of Enpp1 for the ATP substrate. Unlike Enpp2, the SMB- identity, rmsd = 1.1 Å for 341 Cα atoms) and XaNPP (28) (PDB ID like domains are disordered and do not interact with the catalytic 2GSU, rmsd = 1.6 Å for 339 Cα atoms) (Fig. 2 B and C and Fig. S2). domain in Enpp1, suggesting that the SMB-like domains in As in Enpp2 and XaNPP, two zinc ions are bound within the active Enpp1 and Enpp2 have distinct roles. Structural mapping of site of Enpp1. One zinc ion is coordinated by Asp358, His362, and disease-associated mutations indicated the functional signifi- His517, and the other is coordinated by Asp200, Thr238, Asp405, fi cance of the interaction between the catalytic and nuclease-like and His406 (Fig. 3). A previous mutational analysis con rmed the fi domains in both Enpp1 and Enpp2. functional signi cance of these zinc-coordinating residues (29). The α-phosphate group of AMP is bound between the two zinc Results ions, consistent with our functional data showing that Enpp1 A Substrate Specificity. The extracellular region (residues 92–905) of hydrolyzes ATP to produce AMP and PPi (Fig. 1 ). − mouse Enpp1 was overexpressed in HEK293S GnT1 cells as a secreted protein and was purified by P20.1 antibody affinity and Nuclease-Like Domain. The nuclease-like domain of Enpp1 is fi structurally similar to that of Enpp2 (25, 26) (PDB ID 3NKM, 42% gel ltration chromatographies. When ATP was incubated with = α B C the purified protein, the production of AMP and PPi, but not sequence identity, rmsd 1.4 Å for 248 C atoms) (Fig. 2 and and Fig. S3). As in Enpp2, a calcium ion is coordinated by the side ADP and Pi, was detected by mass spectrometry (Fig. 1A), in- chains of Asp780, Asp782, Asp784, and Asp788 and the main- dicating that Enpp1 hydrolyzes the phosphodiester bond between chain carbonyl group of Arg786, forming an EF hand-like motif. In the α- and β-phosphate groups of ATP. A kinetic analysis showed −1 Enpp2, Asp735 (corresponding to Asp780 in Enpp1) interacts with that Enpp1 preferably hydrolyzes ATP (kcat = 16 s , Km = 46 −1 Lys430 (corresponding to Lys479 in Enpp1), and the K430A mu- μM), compared with UTP (kcat = 200 s , Km = 4.3 mM), GTP k = −1 K = k = −1 K = tation impaired the protein stability (26). In Enpp1, Asp780 ( cat 820 s , m 4.2 mM) and CTP ( cat 8.7 s , m 1.2 interacts with Lys479 in the catalytic domain, suggesting the im- mM) (Fig. S1). We also examined the substrate specificity by p ′ p portance of the EF hand-like motif for the interdomain inter- measuring the -nitrophenyl thymidine 5 -monophosphate ( NP- actions in both Enpp1 and Enpp2. TMP)–hydrolyzing activity in the presence of different NMPs. p – AMP inhibited the NP-TMP hydrolyzing activity more potently SMB-Like Domains. Unexpectedly, electron densities were not ob- B than TMP, GMP, and CMP (Fig. 1 ). These results showed that served for the two SMB-like domains, and there is sufficient room to Enpp1 preferably hydrolyzes ATP to produce AMP and PPi and accommodate the two SMB-like domains in the crystal lattice. An confirmed that Enpp1 negatively regulates bone mineralization by SDS/PAGE analysis of the dissolved crystals revealed a single band hydrolyzing ATP, an abundant extracellular nucleotide. (∼100 kDa) similar to the purified protein (Fig. S4A), indicating that the crystallized proteins contain the SMB-like domains. The Overall Architecture. We solved the crystal structures of the ex- reported Enpp1 SMB1 domain (PDB ID 2YS0) is structurally tracellular domain of Enpp1 in complex with four different NMPs similar to the Enpp2 SMB1 domain (25, 26) (PDB ID 3NKM, 53% – (AMP, TMP, GMP, and CMP) at resolutions of 2.7 3.2 Å (Table sequence identity, rmsd = 1.2 Å for 34 Cα atoms) (Fig. S4B). The S1). Because these four crystal structures are essentially identical SMB2 domains of Enpp1 and Enpp2 share 48% sequence identity, (rmsd values less than 0.2 Å for aligned Cα atoms), we describe indicating that the Enpp1 SMB2 domain also adopts a rigid struc- the AMP complex structure, unless otherwise stated. The structure. A structural comparison of Enpp1 with Enpp2 indicated that ture consists of a catalytic domain (residues 190–578), a nuclease- the SMB1 domain of Enpp1 cannot interact with the catalytic do- like domain (residues 629–902), and two linker regions, L1 and main in the way observed in Enpp2 because of steric clashes with the L2 (residues 170–189 and 579–628, respectively) (Fig. 2 A and B). insertion loop (Fig. S4C). In addition, Arg283, Gln290, and Gln344 The catalytic domain interacts with the nuclease-like domain, and in the catalytic domain of Enpp2, which participate in the in- the L2 linker connects the two domains (Fig. 2B). The structure teraction with the SMB2 domain (25), are replaced with Glu330, revealed that Enpp1 is N-glycosylated at Asn267, Asn323, and Glu337, and Asp391, respectively, in Enpp1 (Fig. S4C). These Asn567 and that the domain interaction is reinforced by the observations suggested that, unlike Enpp2, the spatial arrangement Asn567-linked glycan and the Cys462–Cys846 disulfide linkage, of the SMB-like domains of Enpp1 is not fixed by the interaction which correspond to the Asn524-linked glycan and the Cys413– with the catalytic domain. To confirm this idea, we prepared an Cys801 disulfide linkage, respectively, in mouse Enpp2 (Fig. 2 B Enpp1 mutant bearing the Turbo3C protease recognition sequence and C) (25, 26). The spatial arrangement of the catalytic and between SMB2 (Lys169) and L1 (Lys170), incubated the purified nuclease-like domains is conserved in Enpp1 and Enpp2, mutant protein with the protease, and then performed a pulldown

A B C ATP D LysoPLD pNP-TMP pNP-TMP ) ) ) 0.06 15

100 ) –1 –1 –1

10 –1

h min min min

–1 0.04 10 –1 –1 –1 g g g g 50 μ (%) μ μ 5 μ 0.02 5 p NP-TMP– (nmol Relative activity LysoPLD activity (nmol (nmol (nmol 0 0 Hydrolytic activity 0 0 hydrolyzing activity

Nucleotide production AMP ADP No AMP CMP GMP TMP UMP WT F239A H242L D308A Y322A WT

Fig. 1. Biochemical characterization. (A) Enpp1 hydrolyzes ATP to produce AMP and PPi. Puriﬁed Enpp1 was incubated with ATP, and then the reaction products were quantiﬁed by mass spectrometry. (B) Inhibition of Enpp1 activity by NMPs. The enzymatic activity was measured in the absence or presence of 0.5 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–hydrolyzing activities of the wild type and ΔIL mutant of Enpp1. Data are shown as mean ± SD (n = 3).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1208017109 Kato et al. Downloaded by guest on October 1, 2021 A Mouse Enpp1 Crystallization construct Disordered Catalytic domain Nuclease-like domain

Cytosolic TM SMB1 SMB2 L1 IL L2 domain 1 59 82 124 169 190 304 323 579 629 EF hand 905

2+ 2+ B Cys462-Cys846 2 x Zn AMP C Cys413-Cys801 Asn410- 2 x Zn LPA glycan SMB1

SMB2 SMB2 L1 L1

Asn323- glycan Ca2+ Ca2+ SMB1

L2 L2 Asn524- EF hand Asn567- Asn267- insertion loop EF hand glycan glycan glycan Asn53- glycan Nuclease-like domain Catalytic domain Nuclease-like domain Catalytic domain

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 are shown as green and yellow sticks, respectively. The bound zinc and calcium ions are shown as gray and yellow-green spheres, respectively. Disulﬁde linkages are shown as sticks. The two SMB-like domains, which were disordered in the crystal structure, are indicated by circles. (C) Crystal structure of Enpp2 in

complex with 14:0-LPA (PDB ID 3NKN); color code as in B. The SMB1 and SMB2 domains are colored orange and brown, respectively. BIOCHEMISTRY

assay using P20.1-Sepharose. We found that the SMB-like domains 3A) in the AMP complex, and the D308A mutant exhibited de- were not pulled down together with the rest of the protein, sug- creased ATP–hydrolyzing activity (Fig. 1C). In contrast, the gesting that the SMB-like domains do not interact with the catalytic nucleobase moieties of TMP, GMP, and CMP are not recognized domain (Fig. S4D). In contrast, the equivalent Enpp2 mutant with by Enpp1 through hydrogen-bonding interactions (Fig. 3 B–D). the protease recognition sequence between SMB2 (Glu140) and L1 These observations can explain the preference of Enpp1 for ATP (Ser141) was not expressed in HEK293T cells, suggesting differ- as a substrate, as described above (Fig. 1A). Asp308 of Enpp1 is ences in the ﬂexibility between SMB2 and L1 in Enpp1 and Enpp2. replaced with Glu160 in XaNPP (Fig. S2C) (28), suggesting dif- Notably, the protease treatment had almost no effect on the enzy- ferences in the substrate preferences of Enpp1 and XaNPP (al- matic activity (Fig. S4E), indicating that the SMB-like domains are though the substrate preference of XaNPP remains unclear). dispensable for the enzymatic activity of Enpp1. Given that Enpp1 is a type II transmembrane protein, the mobile SMB-like domains of Molecular Determinants of Substrate Speciﬁcity. The present structure Enpp1 may act as a molecular anchor that connects the trans- revealed that the insertion loop (residues 304–323) participates in membrane region and the catalytic domain. the formation of the substrate-binding pocket, with Trp304 in the WPG motif forming a hydrophobic core with Leu196, Ser198, Nucleotide Recognition. The crystal structures in complex with the His242, Ile245, Val246, Trp289, and Thr351 (Fig. 4 A and B). The four different NMPs revealed that the phosphate groups and ri- main-chain amide groups of Trp304 and Tyr322 hydrogen bond bose moieties are recognized by the protein in a similar manner, with the side chains of Ser307 and Asp308, respectively. His242 in and the nucleobase moieties are sandwiched between the side Enpp1 corresponds to Leu213 in Enpp2, which participates in the chains of Phe239 and Tyr322 (Fig. 3). The F239A and Y322A formation of the lipid-binding hydrophobic pocket (Fig. 4 C and mutants showed reduced hydrolytic activities for ATP and pNP- D). As previously reported (30), the H242L mutant showed re- TMP (Fig. 1C). The AMP N6 atom is recognized by Trp304 and duced phosphodiesterase activity (Fig. 1C), indicating the im- Asp308 through a water-mediated hydrogen bond network (Fig. portance of the interaction between His242 and Trp304 in the

ABCD Asp200 Asp200 Asp358 Asp200 Asp200 Asp358 Asp358 Asp358 Asp405 Tyr322 Asp405 Tyr322 Asp405 Asp405 Tyr322 AMP TMP His362 GMP Tyr322 CMP His362 Trp304 His362 His362

H O 2 Asp308 Asp308 Asp308 Thr238 Thr238 Thr238 Asp308 Thr238 His517 His517 His517 His517 Phe239 Phe239 Phe239 His406 His406 His406 Phe239 His406 Asn259 Asn259 Asn259 Asn259

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–FC omit 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.

Kato et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 A B Ala334 Ala334 Arg331 Arg331

Insertion loop Asn323 Asn323 Asn323 Trp338 Trp338 Tyr302 Tyr302 Tyr322 Phe303 Tyr322 Phe303 Leu196 Leu196 Tyr322 AMP Asp308 AMP Asp308 Ser198 Ser198 Thr351 Thr351 Trp304 2+ 2+ AMP Zn Zn Zn2+ His242 Trp304 Ser307 Zn2+ His242 Trp304 Ser307 Phe349 Phe349 Pro305 Pro305 Pro305 Gly306 Trp289 Gly306 Trp289 Phe239 Phe239 Val246 Ile288 Val246 Ile288 Ile245 Ile245 Met555 Met555

C D

SMB1 Hydrophobic pocket

Ala304 Ala304 LPA Ser169 LPA Ser169 Zn2+ Ile167 Phe273 Zn2+ Ile167 Phe273 Zn2+ Zn2+ LPA Leu213 Val302 Leu213 Val302

Trp260 Trp260 Met512 Met512 Phe210 Leu259 Phe210 Leu259 Leu216 Ala217 Leu216 Ala217

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.

formation of the nucleotide-binding pocket. To further examine Discussion whether the insertion loop is the major determinant of the sub- Although Enpp1 hydrolyzes various nucleotide substrates in vitro strate specificity, we prepared the Enpp1 mutant lacking residues (31), its substrate preference was unknown. Our functional analysis 304–323 (ΔIL mutant) and measured its pNP-TMP– and LPC– revealed that Enpp1 preferentially hydrolyzes ATP to produce hydrolyzing activities. The ΔIL mutant showed drastically re- AMP and PPi. Moreover, the present structures provide a molec- duced pNP-TMP–hydrolyzing activity compared with the wild ular basis for PPi production by Enpp1, through ATP hydrolysis. In type (Fig. 1D), indicating the importance of the insertion loop Enpp2, lipid substrates are accommodated in a deep, hydrophobic for the nucleotide recognition. Notably, the ΔIL mutant dis- pocket (25, 26). In contrast, in Enpp1, the nucleotide substrates are played lysoPLD activity (Fig. 1D), suggesting that a lipid-bind- accommodated in the pocket formed by the insertion sequence, ing pocket is generated by the deletion of the insertion loop. which occludes the hydrophobic pocket. These structural differ- However, the lysoPLD activity of the ΔIL mutant (0.025 nmol ences clearly explain why Enpp1 hydrolyzes nucleotides but not − − − − μg 1 h 1) was much lower than that of Enpp2 (38 nmol μg 1 h 1) lipids. Moreover, the deletion of the insertion sequence of Enpp1 (25). In Enpp2, the hydrophobic pocket is formed by conserved resulted in the generation of lysoPLD activity (albeit lower than that hydrophobic residues, such as Ile167, Leu213, Leu216, Ala217, of Enpp2). A structural comparison of Enpp1 and Enpp2 suggested that Enpp2 gained the hydrophobic pocket during the course of Leu259, Trp260, Phe273, Val302, Ala304, and Met512 (25), evolution through the deletion of the insertion loop, followed by which correspond to Leu196, His242, Ile245, Val246, Ile288, amino acid replacements. These observations reinforced our pre- Trp289, Tyr302, Phe349, Thr351, and Met555, respectively, in fi B D vious proposal that the insertion sequence participates in de ning Enpp1 (Fig. 4 and ). Thus, the replacements of His242, the substrate specificity of the Enpp family proteins. Val246, Phe349, and Thr351 in Enpp1 with smaller hydrophobic A number of genetic mutations in Enpp1 are associated with residues may be required for the formation of a hydrophobic GACI, a human disorder with a hypermineralization phenotype pocket optimized for accommodating lipid substrates. In addition, (5–7). Most of these mutations mapped to the catalytic and nu- Tyr302 of Enpp1 is located at a different position from the cor- clease-like domains (Fig. 5 A and B). Among these, the R456Q, responding Phe273 of Enpp2, and Tyr302 interacts with Phe303, L579F, L611V, C726R, N792S, E893X, and Y901S mutations Arg331, Ala334, and Trp338 (Fig. 4B). Taken together, these abolished the enzymatic activity in human Enpp1 (5, 32). The observations suggested that, in addition to the loop deletion, structure of mouse Enpp1 revealed that most of these residues amino acid replacements may have been necessary for the mo- participate in intradomain or interdomain interactions (Fig. 5 B– lecular evolution of Enpp1 to Enpp2. G). In the catalytic domain, Arg438 (Arg456; equivalent residues

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1208017109 Kato et al. Downloaded by guest on October 1, 2021 AB

Human Enpp1 P250L Y312X G342V R481W H500P Y570C G242F P305T R349K R476X S504R G568stop Y659C H777R D804H C C126R S216Y S287F Y371F Y471C Y513C (in mouse) E668K R774C R821P R888W

Cytosolic TM SMB1 SMB2 L1 IL Catalytic domain L2 Nuclease-like domain G domain F 1 77 100 142 187 208 597 648 EF hand 925 Y252Δ Y261X R456Q L579F L611V C726R N792S E893X Y901S G266V (Arg438) (Leu561) (Leu593) (Cys706) (Asn772) (Glu873) (Tyr881) D276N E D CD E FG Leu593 Ca2+ Asp784 Glu589 Cys607 Asp780 Glu490 Glu873 Leu653 Arg641 Cys706 Lys479 His482 Thr291 Lys895 Phe880 Tyr250 Arg438 Ala292 Asp871 Asp258 Gln295 Tyr881 Val297 Glu547 Glu873 Arg868 Tyr552 Leu561 Leu559 Asn772 Ser833 Thr676 Thr816 Phe880 Lys895

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 Enpp1 corresponding to the disease-associated residues of human Enpp1 are shown as gray sticks in B–G. BIOCHEMISTRY of human Enpp1 are indicated in parentheses) hydrogen bonds mutant protein inhibited insulin signaling more efficiently, by with Asp258 and Glu490 (Fig. 5C), and Leu561 (Leu579) forms interacting with the insulin receptor more strongly than the wild a hydrophobic core with Thr291, Ala292, Gln295, Val297, and type (17). Lys173 is replaced by a histidine residue in mouse Leu559 (Fig. 5D). The D276N mutation in Aps276 (equivalent to Enpp1, suggesting that the K173Q polymorphism is specificto Asp258 of mouse Enpp1) also is associated with GACI. In the human Enpp1. Lys173 of human Enpp1 is located within the SMB2 nuclease-like domain, Glu873 (Glu893) interacts with Lys895 and domain, and thus this domain may participate in the interaction Phe880 (Fig. 5E). Asn772 (Asn792) and Tyr881 (Tyr901) hydrogen with the insulin receptor. The SMB2 domain of Enpp2 binds to bond with Thr676 and with Thr816 and Ser833, respectively (Fig. integrins (26), and the SMB domain of vitronectin binds to plas- 5E). These observations suggested that these inactivating muta- minogen activator inhibitor-1 (PAI-1) (33) and the urokinase re- tions result in either the loss of interactions or steric clashes within ceptor (uPAR) (34). Lys173 of human Enpp1 corresponds to the individual domains, highlighting the functional significance of Arg126 of the mouse Enpp2 SMB2 domain and Tyr28 of the the catalytic and nuclease-like domains. The ttw mouse carries the vitronectin SMB domain (Fig. S4B). Arg126 of Enpp2 is exposed G568stop nonsense mutation, which results in the deletion of the to the solvent (25), and Tyr28 of vitronectin participates in the nuclease-like domain (4), and the Enpp1 mutant protein with interaction with PAI-1 (33) and uPAR (34). These observations a truncated nuclease-like domain (Δ804–905) is catalytically in- support the notion that Lys173 of human Enpp1 participates in the active (18), further highlighting the functional significance of the interaction with the insulin receptor. nuclease-like domain. Leu593 (Leu611) on the L2 linker interacts We previously hypothesized that the interaction between the with Arg641 and Leu653 in the nuclease-like domain, and Arg641 catalytic and nuclease-like domains contributes to maintaining the interacts with Glu589 on the L2 linker (Fig. 5E). Cys706 (Cys726) structural integrity of the hydrophobic pocket in Enpp2 (25). in the nuclease-like domain forms a disulfide bond with Cys607 on However, the present results revealed that the interdomain in- the L2 linker (Fig. 5F). These observations indicated that the interaction is conserved in Enpp1 and is important for the enzymatic teraction between the nuclease-like domain and the L2 linker is activity, although Enpp1 lacks a hydrophobic pocket. Recent mo- important for the enzymatic activity. In addition, disease-causing lecular dynamics simulations revealed that the interdomain in- mutations are mapped at the domain interface (H500P in the teraction contributes mainly to the correct positioning of the catalytic domain and D804H and R888W in the nuclease-like catalytic threonine residue in the catalytic domain, explaining the domain) (Fig. 5G). His482 (His500) and Arg868 (Arg888) hydro- requirement of the conserved interdomain interaction in Enpp1 gen bond with Asp871 and with Tyr250 and Glu547, respectively. and Enpp2 (35). We also found that the SMB-like domains of Asp784 (Asp804) is located in the EF hand-like motif, and Asp780 Enpp1 do not interact with the catalytic domain (Fig. 2B) and are within this motif interacts with Lys479 in the catalytic domain. dispensable for the enzymatic activity (Fig. S4E), consistent with These observations indicated the functional significance of the the mapping of most of the disease-causing mutations on the cat- interaction between the catalytic and nuclease-like domains. The alytic and nuclease-like domains (Fig. 5A)(5–7). These observa- structural mapping of the disease-causing mutations revealed that tions suggested that the SMB-like domains of Enpp1 act as the integrity not only of the individual domains but also of the a flexible molecular anchor, consistent with the notion that the interdomain interactions is important for the enzymatic activity SMB2 domain of human Enpp1 interacts with the insulin receptor. of Enpp1. In Enpp2, the SMB-like domains interact with the catalytic domain Enpp1 interacts directly with the insulin receptor and prevents and form a hydrophobic channel, which may serve as an exit for insulin-induced conformational changes in the receptor, thereby lipid products to specific G protein-coupled receptors (25, 26). inhibiting insulin signaling (14). The Enpp1 K173Q polymorphism Thus, the distinct arrangements of the SMB-like domains are likely is associated with obesity and type 2 diabetes (15), and the K173Q to reflect their functional differences in Enpp1 and Enpp2.

Kato et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 In summary, our findings suggest that Enpp1 participates in collection and refinement statistics are provided in Table S1. Molecular different biological processes through distinct sets of domains: the graphics were prepared using CueMol (http://www.cuemol.org). Detailed catalytic and nuclease-like domains for bone mineralization and methods are described in SI Materials and Methods. the SMB-like domains for insulin signaling. Moreover, our findings indicate that Enpp1 and Enpp2 exert diverse cellular func- ACKNOWLEDGMENTS. We thank the beamline staff at BL32XU at SPring-8 for technical help during data collection. This work was supported by grants tions because of their distinct domain arrangements and active- from the Japan Society for the Promotion of Science (JSPS) through its Fund- site architectures, although they share similar primary structures. ing Program for World-Leading Innovative Research and Development on Science and Technology (FIRST) program (to O.N.); from the Japan Science Materials and Methods and Technology Agency (JST) through the Core Research for Evolutional The extracellular region (residues 92–905) of mouse Enpp1 was expressed, Science and Technology (CREST) program on the Creation of Basic Medical Technologies to Clarify and Control the Mechanisms Underlying Chronic In- purified, and crystallized as described previously (36). X-ray diffraction data flammation (to J.A. and O.N.); by a Grant-in-Aid for Scientific Research on were collected at 100 K on beamline BL32XU at SPring-8 (Hyogo, Japan). The Innovative Areas from the Ministry of Education, Culture, Sports, Science and crystal structure in complex with AMP was determined by the single-wave- Technology (MEXT) (to R.I. and O.N.); by a Grant-in-Aid for Young Scientists length anomalous dispersion method, and the crystal structures in complex (A) from MEXT (to H.N.); and by a Grant-in-Aid for Scientific Research (S) with the other NMPs were determined by molecular replacement. Data from MEXT (to O.N.).

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