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Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor

Nature volume 426, pages 525–530 (2003)Cite this article

Abstract

Small GTP-binding (G) proteins are activated by GDP/GTP nucleotide exchange stimulated by guanine nucleotide exchange factors (GEFs). Nucleotide dissociation from small G protein–GEF complexes involves transient GDP-bound intermediates whose structures have never been described. In the case of Arf proteins, small G proteins that regulate membrane traffic in eukaryotic cells, such intermediates can be trapped either by the natural inhibitor brefeldin A or by charge reversal at the catalytic glutamate of the Sec7 domain of their GEFs. Here we report the crystal structures of these intermediates that show that membrane recruitment of Arf and nucleotide dissociation are separate reactions stimulated by Sec7. The reactions proceed through sequential rotations of the Arf·GDP core towards the Sec7 catalytic site, and are blocked by interfacial binding of brefeldin A and unproductive stabilization of GDP by charge reversal. The structural characteristics of the reaction and its modes of inhibition reveal unexplored ways in which to inhibit the activation of small G proteins.

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Figure 1: Snapshots of the nucleotide exchange reaction.
Figure 2: The Arf-Sec7 interfacial platform.
Figure 3: Interfacial inhibition of nucleotide exchange by BFA.
Figure 4: Stabilization of GDP by charge reversal at the glutamic finger of the Sec7 domain.

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Acknowledgements

We thank the ESRF for provision of synchrotron radiation facilities and the staff of beamlines BM30A, ID14-EH2 and ID14-EH3 for assistance. This work was supported by grants from the Association pour la Recherche contre le Cancer, the CNRS, the Ministère de la Recherche and the Human Frontier Science Program. We thank B. Antonny and co-workers at the IPMC (CNRS) for the gift of the plasmids used in this work and for discussions. The Arf1(Δ17)–Gea2-Sec7 coordinates are a gift from J. Goldberg.

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  1. Laboratoire d'Enzymologie et Biochimie Structurales, CNRS UPR 9063, Avenue de la Terrasse, 91198, Gif sur Yvette, France

    Louis Renault, Bernard Guibert & Jacqueline Cherfils

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Correspondence to Jacqueline Cherfils.

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Renault, L., Guibert, B. & Cherfils, J. Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor. Nature 426, 525–530 (2003). https://doi.org/10.1038/nature02197

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