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Structural basis of substrate discrimination and integrin binding by autotaxin

Abstract

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B–like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

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Figure 1: The structure of ATX.
Figure 2: ATX domains and closest structural homologs shown in matched orientations.
Figure 3: The ATX catalytic site and substrate binding pocket.
Figure 4: Biochemical analysis of human ATX and mutants.
Figure 5: The SMB domains and integrin-mediated interaction with activated platelets.

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Acknowledgements

Crystallographic experiments were conducted at the PX beamline at the Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland; at the European Synchrotron Radiation Facility beamline ID23-2; at the Diamond Light Source ID19 microfocus beamline; and at the GM/CA-CAT, NE-CAT and LS-CAT beamlines at the Advanced Photon Source. We thank all beamline scientists and especially S. McSweeney, D. Flo, K. Schultze-Briese, T. Tomizaki, G. Evans and J. Grimes for data collection assistance. This research was supported by grants from the Dutch Cancer Society to W.H.M., the US National Institutes of Health to A.J.M. (GM50388, P20RR021954), C.V.K. (GM094155) and S.S.S. (HL078663), F30 HL099272 to Z.F. and an American Heart Association postdoctoral fellowship to T.W. Material for these authors is the result of work supported with the resources and use of the facilities at the Lexington Veterans Affairs Medical Center. E.C. and A.P. thank V. De Marco, D. Littler and P. Rucktooa for assistance with crystal mounting and data collection. J.H. and A.P. thank T. Walter, P. Celie, M. Sturnaiolo and T. Heidebrecht for advice and assistance, the Netherlands Cancer Institute and Pfizer for jointly providing a pre-doctoral fellowship to J.H., T. Sixma for feedback on the manuscript and the NKI Protein Facility for infrastructure access.

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Contributions

J.H. purified, crystallized and collected diffraction data for native crystal forms; established co-crystals, collected diffraction data and participated in structure determination of the HA155 complex; and cloned, expressed and assayed activity of catalytic and binding site mutants. S.K. directly supervised Pfizer scientists, collected and processed diffraction data, determined the first structure, built and refined crystal structures; E.C. established cell lines, protein expression and purification, crystallization protocols for the first native crystal form, and collected diffraction data; J.E.D. identified and produced the second, high resolution, native crystal form and collected diffraction data; T.W. and Z.F. generated all reagents for ATX platelet binding experiments and did the relevant assays; L.A.v.M. participated in protein expression and with the help of H.M.H.G.A. and A.J.S.H., established ATX assays; H.M.H.G.A. made the HA155 inhibitor; L.v.Z. conducted the PAI-1 experiment; S.J. generated the rat ATX mutant used for crystallization, M.A. did the EF hand–like mutagenesis experiments and assays; T.H. purified protein; L.E.P. expressed protein; T.E.B. coordinated the Pfizer scientists; K.H. assisted with crystal growth, manipulation and data collection; M.K. helped establish the stable cell line producing rat ATX; C.W.V.K., S.S.S. and A.J.M. designed and supervised platelet binding experiments; H.O. supervised inhibitor design at the NKI site; M.B. supervised EF-hand experiments and participated in project coordination; W.H.M. and A.P. initiated and coordinated the project and supervised experiments at the NKI. A.P. also determined and refined crystal structures, prepared all display items and wrote the paper, with input from W.H.M., S.K., M.B. and A.J.M.

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Correspondence to Andrew J Morris, Wouter H Moolenaar or Anastassis Perrakis.

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Competing interests

At the time this research was performed, S.K., J.E.D., T.H., L.E.P. and T.E.B. were employees of Pfizer, Inc.

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Hausmann, J., Kamtekar, S., Christodoulou, E. et al. Structural basis of substrate discrimination and integrin binding by autotaxin. Nat Struct Mol Biol 18, 198–204 (2011). https://doi.org/10.1038/nsmb.1980

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