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Crystal structure of human CDC7 kinase in complex with its activator DBF4

Nature Structural & Molecular Biology volume 19, pages 1101–1107 (2012)Cite this article

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Abstract

CDC7 is a serine/threonine kinase that is essential for the initiation of eukaryotic DNA replication. CDC7 activity is controlled by its activator, DBF4. Here we present crystal structures of human CDC7–DBF4 in complex with a nucleotide or ATP-competing small molecules, revealing the active and inhibited forms of the kinase, respectively. DBF4 wraps around CDC7, burying approximately 6,000 Å2 of hydrophobic molecular surface in a bipartite interface. The effector domain of DBF4, containing conserved motif C, is essential and sufficient to support CDC7 kinase activity by binding to the kinase N-terminal lobe and stabilizing its canonical αC helix. DBF4 motif M latches onto the C-terminal lobe of the kinase, acting as a tethering domain. Our results elucidate the structural basis for binding to and activation of CDC7 by DBF4 and provide a framework for the design of more potent and specific CDC7 inhibitors.

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Figure 1: Schematic of CDC7 and DBF4 proteins and the enzymatic activity of a representative set of deletion constructs.
Figure 2: Structure of the CDC7–DBF4 heterodimer and a view on the active site.
Figure 3: Details of the DBF4 structure and its interface with CDC7.
Figure 4: DBF4 motif C and its contacts with αC are pivotal for the ability of DBF4 to activate CDC7.
Figure 5: The inward face of CDC7 αC becomes exposed to solvent in the absence of DBF4 motif C.
Figure 6: Binding of ATP-competitive inhibitors to the CDC7 active site.

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  • 05 December 2012

    In the version of this supplementary file originally posted online, there were partial omissions of data in graphs and chromatograms on pages 2, 4 and 8. These errors have been corrected in this file as of 5 December 2012.

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Acknowledgements

We thank J. Moore for expert help in crystallization screening, J.F. Diffley, G.N. Maertens and V. Pye for critical reading of the manuscript and the staff of Diamond beamlines I02, I03 and I04 for assistance in data collection. This work was supported by UK Medical Research Council grant G0900116 (P.C.) and intramural funding from Cancer Research UK (P.C.).

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Authors and Affiliations

  1. Cancer Research UK, London Research Institute, Clare Hall Laboratories, Potters Bar, UK

    Siobhan Hughes, Andrea Di Fonzo, Ambrosius P Snijders & Peter Cherepanov

  2. Cancer Research Technology Discovery Laboratories, Wolfson Institute for Biomedical Research, London, UK

    Frédéric Elustondo, Frédéric G Leroux & Ai C Wong

  3. Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, UK

    Stephen J Matthews

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Contributions

S.H. optimized expression constructs, purified proteins and assessed their in vitro kinase activities. S.H. and P.C. carried out crystallization screening, prepared crystals, collected X-ray diffraction data and solved and refined the structures. F.E., F.G.L. and A.C.W. evaluated small molecules using in vitro kinase assays. S.J.M. acquired and analyzed NMR spectra. A.D.F. and A.P.S. performed mass spectrometry analyses. P.C. and S.H. wrote the paper.

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Correspondence to Peter Cherepanov.

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The authors declare no competing financial interests.

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Supplementary Figures 1–6 and Supplementary Table 1 (PDF 6407 kb)

Supplementary Video 1

Overall structure of the CDC7–DBF4 complex. (MOV 3990 kb)

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Hughes, S., Elustondo, F., Di Fonzo, A. et al. Crystal structure of human CDC7 kinase in complex with its activator DBF4. Nat Struct Mol Biol 19, 1101–1107 (2012). https://doi.org/10.1038/nsmb.2404

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