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G domain dimerization controls dynamin's assembly-stimulated GTPase activity

Nature volume 465, pages 435–440 (2010)Cite this article

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Abstract

Dynamin is an atypical GTPase that catalyses membrane fission during clathrin-mediated endocytosis. The mechanisms of dynamin’s basal and assembly-stimulated GTP hydrolysis are unknown, though both are indirectly influenced by the GTPase effector domain (GED). Here we present the 2.0 Å resolution crystal structure of a human dynamin 1-derived minimal GTPase–GED fusion protein, which was dimeric in the presence of the transition state mimic GDP.AlF4-.The structure reveals dynamin’s catalytic machinery and explains how assembly-stimulated GTP hydrolysis is achieved through G domain dimerization. A sodium ion present in the active site suggests that dynamin uses a cation to compensate for the developing negative charge in the transition state in the absence of an arginine finger. Structural comparison to the rat dynamin G domain reveals key conformational changes that promote G domain dimerization and stimulated hydrolysis. The structure of the GTPase–GED fusion protein dimer provides insight into the mechanisms underlying dynamin-catalysed membrane fission.

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Figure 1: Structure of GG dimer.
Figure 2: Catalytic machinery involved in dynamin GTP hydrolysis.
Figure 3: Active site conformational changes induced by dynamin G-domain dimerization and GTP hydrolysis.
Figure 4: Functional analyses of dynamin active site mutants.
Figure 5: Structure and conformational changes of the BSE.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates of the GG long-axis and short-axis dimers are deposited in the Protein Data Bank with accession numbers 2X2E and 2X2F, respectively.

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Acknowledgements

We thank V. Lukiyanchuk for assistance in cloning and purification; A. Hickman, J. Mindell and R. Ramachandran for discussions and technical advice; T. Pucadyil and R. Ramachandran for critical reading of the manuscript; J. Hinshaw and J. Mears for providing the unpublished coordinates for the GTPase docking model derived from their cryo-EM docking studies; and R. Stevens and I. Wilson for structural advice, guidance and the use of their laboratory facilities in the early stages of this work. This work was supported by NIH grants GM42455 and MH61345 (to S.L.S.) and the Intramural Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the NIH. J.S.C. was supported by a Ruth Kirschstein individual predoctoral fellowship from the NIMH (MH081419) and by a postdoctoral Intramural Research Training Award from NIDDK. Data were collected at the SER-CAT 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Use of the APS was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no. W-31-109-Eng-38.

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Authors and Affiliations

  1. Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20892, USA ,

    Joshua S. Chappie & Fred Dyda

  2. Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA,

    Joshua S. Chappie, Sharmistha Acharya, Marilyn Leonard & Sandra L. Schmid

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  1. Joshua S. Chappie
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Contributions

J.S.C. designed, purified, and characterized all GG proteins and crystallized the GG dimer; J.S.C. and F.D. collected X-ray diffraction data and solved the structures; S.A. and M.L. purified full-length dynamin constructs and carried out biochemical assays; J.S.C., S.L.S. and F.D. designed experiments and interpreted data; and J.S.C, S.L.S. and F.D. prepared the manuscript.

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Correspondence to Sandra L. Schmid or Fred Dyda.

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The authors declare no competing financial interests.

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Chappie, J., Acharya, S., Leonard, M. et al. G domain dimerization controls dynamin's assembly-stimulated GTPase activity. Nature 465, 435–440 (2010). https://doi.org/10.1038/nature09032

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