Article

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

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20892, USA.
Nature (Impact Factor: 41.46). 05/2010; 465(7297):435-40. DOI: 10.1038/nature09032
Source: PubMed

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 A 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.AlF(4)(-).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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    • "The α2 helix makes a direct intermolecular contact with the BSE of neighbouring dynamin molecules within the tetramer, forming a 'dimer-dimer' interface referred to as interface 5[2,13]. The BSE itself has also been implicated as a remarkably dynamic domain that appears to respond to the catalytic cycle of the G domain[44,45]. We propose that the BSE may also respond to the binding state of the PRD via the dimer-dimer interface in order to allosterically regulate G domain activity. "
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    ABSTRACT: Dynamin I is a highly regulated GTPase enzyme enriched in nerve terminals which mediates vesicle fission during synaptic vesicle endocytosis. One regulatory mechanism involves its interactions with proteins containing Src homology 3 (SH3) domains. At least 30 SH3 domain-containing proteins bind dynamin at its proline-rich domain (PRD). Those that stimulate dynamin activity act by promoting its oligomerisation. We undertook a systematic parallel screening of 13 glutathione-S-transferase (GST)-tagged endocytosis-related SH3 domains on dynamin binding, GTPase activity and oligomerisation. No correlation was found between dynamin binding and their potency to stimulate GTPase activity. There was limited correlation between the extent of their ability to stimulate dynamin activity and the level of oligomerisation, indicating an as yet uncharacterised allosteric coupling of the PRD and G domain. We examined the two variants, dynamin Iab and Ibb, which differ in the alternately splice middle domain α2 helix. They responded differently to the panel of SH3s, with the extent of stimulation between the splice variants varying greatly between the SH3s. This study reveals that SH3 binding can act as a heterotropic allosteric regulator of the G domain via the middle domain α2 helix, suggesting an involvement of this helix in communicating the PRD-mediated allostery. This indicates that SH3 binding both stabilises multiple conformations of the tetrameric building block of dynamin, and promotes assembly of dynamin-SH3 complexes with distinct rates of GTP hydrolysis.
    Full-text · Article · Dec 2015 · PLoS ONE
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    • "In GG1 GDP this interaction is lost due to the different position of switch I. In their analysis of the transition state structure GG GDP.AlFx Chappie and co-workers [10] concluded that interactions of TSL and especially CSL restrict the conformational flexibility of switch II to ensure efficient positioning during catalysis. However, the portion of switch II involved in nucleotide binding and positioning of the catalytic water (residues 136e141) in GG1 GDP does not show greater mobility than its counterparts in GG1 GDP.AlFx or GG1 GppCp , as comparison of the temperature factors indicates (not shown). "
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    ABSTRACT: Dynamin is the prototype of a family of large multi-domain GTPases. The 100 kDa protein is a key player in clathrin-mediated endocytosis, where it cleaves off vesicles from membranes using the energy from GTP hydrolysis. We have solved the high resolution crystal structure of a fusion protein of the GTPase domain and the bundle signaling element (BSE) of dynamin 1 liganded with GDP. The structure provides a hitherto missing snapshot of the GDP state of the hydrolytic cycle of dynamin and reveals how the switch I region moves away from the active site after GTP hydrolysis and release of inorganic phosphate. Comparing our structure of the GDP state with the known structures of the GTP state, the transition state and the nucleotide-free state of dynamin 1 we describe the structural changes through the hydrolytic cycle.
    Full-text · Article · Nov 2015 · Biochemical and Biophysical Research Communications
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    • "Fusion was GTP hydrolysis dependent, prompting the idea that atlastin might represent the long-sought GTP-dependent fusion machinery for the ER (Dreier, 2000). It is now known that atlastin, similar to dynamin (Chappie et al., 2010), and other GTPases that undergo nucleotide-dependent head-to-head dimerization (Gasper et al., 2009), forms a transhomodimer as it catalyzes GTP hydrolysis (Byrnes et al., 2013). Transdimerization in atlastin is further accompanied by a rigid-body rotation of a threehelix bundle (3HB) connecting each GTPase head domain to its membrane anchor. "
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    ABSTRACT: At least 38 distinct missense mutations in the neuronal atlastin1/SPG3A GTPase are implicated in an autosomal dominant form of hereditary spastic paraplegia (HSP), a motor-neurological disorder manifested by lower limb weakness and spasticity and length dependent axonopathy of corticospinal motor neurons. Because the atlastin GTPase is sufficient to catalyze membrane fusion and required to form the ER network, at least in non-neuronal cells, it is logically assumed that defects in ER membrane morphogenesis due to impaired fusion activity are the primary drivers of SPG3A-associated HSP. Here we analyzed a subset of established atlastin1/SPG3A disease variants using cell-based assays for atlastin-mediated ER network formation and biochemical assays for atlastin-catalyzed GTP hydrolysis, dimer formation and membrane fusion. As anticipated, some variants exhibited clear deficits. Surprisingly however, at least two disease variants, one of which represents that most frequently identified in SPG3A HSP patients, displayed wild type levels of activity in all assays. The same variants were also capable of coredistributing ER-localized REEP1, a recently identified function of atlastins that requires its catalytic activity. Altogether, these findings indicate that a deficit in the membrane fusion activity of atlastin1 may be a key contributor, but not required, for HSP causation. © 2015 by The American Society for Cell Biology.
    Full-text · Article · Mar 2015 · Molecular Biology of the Cell
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