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Crystal structure of the integral membrane diacylglycerol kinase

Nature volume 497, pages 521–524 (2013)Cite this article

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Abstract

Diacylglycerol kinase catalyses the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane-derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria1. For half a century, this 121-residue kinase has served as a model for investigating membrane protein enzymology1,2,3,4,5,6, folding7,8, assembly9,10,11,12 and stability1,13. Here we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type. These reveal a homo-trimeric enzyme with three transmembrane helices and an amino-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site that is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are, however, at variance with a published solution NMR model14 in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.

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Figure 1: Crystal structure of Δ4 DgkA.
Figure 2: Rationalizing functional biochemistry with the crystal structure of DgkA.
Figure 3: Putative active site of DgkA complete with lipid and Mg2+-ATP substrates and activating zinc.
Figure 4: Comparison of the crystal and solution NMR structures of DgkA.

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Accession codes

Accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for wild-type, Δ4 and Δ7 DgkA are deposited in the Protein Data Bank under accession codes 3ZE4, 3ZE5 and 3ZE3, respectively.

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Acknowledgements

We acknowledge support from Science Foundation Ireland (grants 07/IN.1/B1836 and 12/IA/1255) and the National Institutes of Health (grants GM75915, P50GM073210, U54GM094599) and FP7 (COST CM0902). We thank C. R. Sanders for providing E. coli strain WH1061 and for his collegiality. The assistance and support of beamline scientists at the Advanced Photon Source (23-ID) and Diamond Light Source (I24) are gratefully acknowledged. We thank R. Sanishvili for assistance with zinc analysis, C. Boland and J. Tan for help with diffraction data collection, and A. Coughlan for help with lipid synthesis.

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Author notes

  1. Valerie E. Pye, David Aragão & Christine Doherty

    Present address: Present addresses: Clare Hall Laboratories, Cancer Research UK, Blanche Lane, South Mimms, Hertfordshire EN6 3LD, UK (V.E.P.); Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria VIC 3168, Australia (D.A.); The Matrix Biology Group, Kennedy Institute of Rheumatology, University of Oxford, 65 Aspenlea Road, Hammersmith, London W6 8LH, UK (C.D.).,

Authors and Affiliations

  1. School of Biochemistry and Immunology & School of Medicine, Trinity College Dublin, Dublin 2, Ireland,

    Dianfan Li, Joseph A. Lyons, Valerie E. Pye, Lutz Vogeley, David Aragão, Syed T. A. Shah, Christine Doherty, Margaret Aherne & Martin Caffrey

  2. CSIR, Biosciences, Meiring-Naude Road, Pretoria 0184, Gauteng, South Africa,

    Colin P. Kenyon

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Contributions

D.L. produced, purified, crystallized and functionally characterized the protein and its variants, collected and processed diffraction data, refined and analysed the structures, and helped write the manuscript. J.A.L., D.A. and V.E.P. collected and processed data, solved, refined and analysed the structures, and helped write the manuscript. L.V. helped process data, solve and analyse structures and write the manuscript. M.A., C.D. and V.E.P. helped with protein and crystal production. C.P.K. performed docking. S.T.A.S. provided 7.8 MAG for crystallization. M.C. was responsible for the overall project strategy and management and oversaw manuscript preparation.

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Correspondence to Martin Caffrey.

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The authors declare no competing financial interests.

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This file contains a Supplementary Discussion, Supplementary Tables 1-2, Supplementary Figures 1-15 and additional references. (PDF 2723 kb)

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Li, D., Lyons, J., Pye, V. et al. Crystal structure of the integral membrane diacylglycerol kinase. Nature 497, 521–524 (2013). https://doi.org/10.1038/nature12179

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