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Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding

Nature Chemical Biology volume 8, pages 111–116 (2012)Cite this article

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Abstract

Inositol pyrophosphates (such as IP7 and IP8) are multifunctional signaling molecules that regulate diverse cellular activities. Inositol pyrophosphates have 'high-energy' phosphoanhydride bonds, so their enzymatic synthesis requires that a substantial energy barrier to the transition state be overcome. Additionally, inositol pyrophosphate kinases can show stringent ligand specificity, despite the need to accommodate the steric bulk and intense electronegativity of nature's most concentrated three-dimensional array of phosphate groups. Here we examine how these catalytic challenges are met by describing the structure and reaction cycle of an inositol pyrophosphate kinase at the atomic level. We obtained crystal structures of the kinase domain of human PPIP5K2 complexed with nucleotide cofactors and either substrates, product or a MgF3− transition-state mimic. We describe the enzyme's conformational dynamics, its unprecedented topological presentation of nucleotide and inositol phosphate, and the charge balance that facilitates partly associative in-line phosphoryl transfer.

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Figure 1: Overall structure of hPPIP5K2KD, a member of the ATP–grasp fold superfamily.
Figure 2: Comparison of substrate binding pockets for hPPIP5K2KD and ITPK1.
Figure 3: Structural and mutagenic analysis of residues in hPPIP5K2KD that participate in substrate binding.
Figure 4: Snapshots of the reaction mechanism.
Figure 5: The relative topologies of nucleotide and substrate in inositol phosphate kinases.
Figure 6: A conserved catalytic blueprint in hPPIP5K2KD.

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Acknowledgements

Data were collected at the Southeast Regional Collaborative Access Team 22-ID/22-BM beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at http://www.ser-cat.org/members.html. The use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38. This work was supported by the Intramural Research Program of the National Institutes of Health and National Institute of Environmental Health Sciences (NIEHS). We are grateful to L.C. Pedersen for advice and support. We also thank H. Ke for his assistance in writing the manuscript. Expression vectors were prepared by the NIEHS Protein Expression Core Facility.

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Authors and Affiliations

  1. Inositol Signaling Group, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

    Huanchen Wang & Stephen B Shears

  2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    J R Falck

  3. Macromolecular Structure Group, Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

    Traci M Tanaka Hall

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  1. Huanchen Wang
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Contributions

H.W., T.M.T.H. and S.B.S. designed experiments and wrote the manuscript. H.W. and S.B.S. performed the experiments. J.R.F. synthesized substrate for hPPIP5K2.

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Correspondence to Huanchen Wang.

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Wang, H., Falck, J., Hall, T. et al. Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding. Nat Chem Biol 8, 111–116 (2012). https://doi.org/10.1038/nchembio.733

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