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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    • "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.
    Molecular Biology of the Cell 03/2015; 26(9). DOI:10.1091/mbc.E14-10-1447 · 4.47 Impact Factor
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    • "GTP, which binds near the GTPase dimer interface, stabilizes the dimer contact, and formation of the interface greatly increases the rate of nucleotide hydrolysis. The GTPase-proximal segment of the stalk, a helical bundle sometimes called the " bundle signaling element " (BSE; Chappie et al., 2009, 2010), responds through the " switch 1 " element of the GTPase to the occupancy of the nucleotide-binding site. Structures that include both the GTPase and the BSE show that the latter has one conformation (relative to the Ras-like domain) in the GTP-bound state (as represented by the nonhydrolyzable analogue, GMPPCP) and a quite different one in the transition state (as represented by GDP- AlF 4− ). "
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    ABSTRACT: Dynamin, the GTPase required for clathrin-mediated endocytosis, is recruited to clathrin-coated pits in two sequential phases. The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit. Using gene-edited cells that express dynamin2-EGFP instead of dynamin2 and live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution, we detected the arrival of dynamin at coated pits and defined dynamin dimers as the preferred assembly unit. We also used live-cell spinning-disk confocal microscopy calibrated by single-molecule EGFP detection to determine the number of dynamins recruited to the coated pits. A large fraction of budding coated pits recruit between 26 and 40 dynamins (between 1 and 1.5 helical turns of a dynamin collar) during the recruitment phase associated with neck fission; 26 are enough for coated vesicle release in cells partially depleted of dynamin by RNAi. We discuss how these results restrict models for the mechanism of dynamin-mediated membrane scission.
    Molecular Biology of the Cell 09/2014; 25(22). DOI:10.1091/mbc.E14-07-1240 · 4.47 Impact Factor
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    • "Dynamin2 is recruited to nascent clathrin-coated pits where it forms a collar-like structure at the neck of deeply invaginated clathrin-coated pits (87–89). GTP-hydrolysis mediated changes in dynamin2 conformation lead to membrane fission and clathrin-coated vesicle release (86, 90–92). Dynamin2 also facilitates membrane binding (93–95) and membrane curvature (96) during CME. "
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    ABSTRACT: Extracellular vesicles (EVs) are small vesicles released by donor cells that can be taken up by recipient cells. Despite their discovery decades ago, it has only recently become apparent that EVs play an important role in cell-to-cell communication. EVs can carry a range of nucleic acids and proteins which can have a significant impact on the phenotype of the recipient. For this phenotypic effect to occur, EVs need to fuse with target cell membranes, either directly with the plasma membrane or with the endosomal membrane after endocytic uptake. EVs are of therapeutic interest because they are deregulated in diseases such as cancer and they could be harnessed to deliver drugs to target cells. It is therefore important to understand the molecular mechanisms by which EVs are taken up into cells. This comprehensive review summarizes current knowledge of EV uptake mechanisms. Cells appear to take up EVs by a variety of endocytic pathways, including clathrin-dependent endocytosis, and clathrin-independent pathways such as caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft–mediated internalization. Indeed, it seems likely that a heterogeneous population of EVs may gain entry into a cell via more than one route. The uptake mechanism used by a given EV may depend on proteins and glycoproteins found on the surface of both the vesicle and the target cell. Further research is needed to understand the precise rules that underpin EV entry into cells.
    08/2014; 3:24641. DOI:10.3402/jev.v3.24641
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