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

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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
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