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• W2058359969 abstract "Numerous vesiculation processes throughout the eukaryotic cell are dependent on the protein dynamin, a large GTPase that constricts lipid bilayers. We have combined X-ray crystallography and cryo-electron microscopy (cryo-EM) data to generate a coherent model of dynamin-mediated membrane constriction. GTPase and pleckstrin homology domains of dynamin were fit to cryo-EM structures of human dynamin helices bound to lipid in nonconstricted and constricted states. Proteolysis and immunogold labeling experiments confirm the topology of dynamin domains predicted from the helical arrays. Based on the fitting, an observed twisting motion of the GTPase, middle, and GTPase effector domains coincides with conformational changes determined by cryo-EM. We propose a corkscrew model for dynamin constriction based on these motions and predict regions of sequence important for dynamin function as potential targets for future mutagenic and structural studies. Numerous vesiculation processes throughout the eukaryotic cell are dependent on the protein dynamin, a large GTPase that constricts lipid bilayers. We have combined X-ray crystallography and cryo-electron microscopy (cryo-EM) data to generate a coherent model of dynamin-mediated membrane constriction. GTPase and pleckstrin homology domains of dynamin were fit to cryo-EM structures of human dynamin helices bound to lipid in nonconstricted and constricted states. Proteolysis and immunogold labeling experiments confirm the topology of dynamin domains predicted from the helical arrays. Based on the fitting, an observed twisting motion of the GTPase, middle, and GTPase effector domains coincides with conformational changes determined by cryo-EM. We propose a corkscrew model for dynamin constriction based on these motions and predict regions of sequence important for dynamin function as potential targets for future mutagenic and structural studies. The formation of transport vesicles during receptor-mediated endocytosis, caveolae internalization, and clathrin-mediated membrane trafficking from the Golgi and recycling endosomes requires the large GTPase dynamin (Hinshaw, 2000Hinshaw J.E. Dynamin and its role in membrane fission.Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (564) Google Scholar, Praefcke and McMahon, 2004Praefcke G.J. McMahon H.T. The dynamin superfamily: universal membrane tubulation and fission molecules?.Nat. Rev. Mol. Cell Biol. 2004; 5: 133-147Crossref PubMed Scopus (1059) Google Scholar). During vesiculation, dynamin wraps around the necks of invaginating pits and plays an active role in the final stages of vesicle fission. In support of this model, dynamin, which exists as a tetramer in solution (Binns et al., 1999Binns D.D. Barylko B. Grichine N. Atkinson M.A. Helms M.K. Jameson D.M. Eccleston J.F. Albanesi J.P. Correlation between self-association modes and GTPase activation of dynamin.J. Protein Chem. 1999; 18: 277-290Crossref PubMed Scopus (45) Google Scholar, Muhlberg et al., 1997Muhlberg A.B. Warnock D.E. Schmid S.L. Domain structure and intramolecular regulation of dynamin GTPase.EMBO J. 1997; 16: 6676-6683Crossref PubMed Scopus (195) Google Scholar), has been shown to polymerize into large oligomeric spirals (Carr and Hinshaw, 1997Carr J.F. Hinshaw J.E. Dynamin assembles into spirals under physiological salt conditions upon the addition of GDP and γ-phosphate analogues.J. Biol. Chem. 1997; 272: 28030-28035Crossref PubMed Scopus (88) Google Scholar, Hinshaw and Schmid, 1995Hinshaw J.E. Schmid S.L. Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding.Nature. 1995; 374: 190-192Crossref PubMed Scopus (642) Google Scholar) that are representative of the dynamin structures observed at the necks of budding vesicles (Evergren et al., 2004Evergren E. Tomilin N. Vasylieva E. Sergeeva V. Bloom O. Gad H. Capani F. Shupliakov O. A pre-embedding immunogold approach for detection of synaptic endocytic proteins in situ.J. Neurosci. Methods. 2004; 135: 169-174Crossref PubMed Scopus (30) Google Scholar, Iversen et al., 2003Iversen T.G. Skretting G. van Deurs B. Sandvig K. Clathrin-coated pits with long, dynamin-wrapped necks upon expression of a clathrin antisense RNA.Proc. Natl. Acad. Sci. USA. 2003; 100: 5175-5180Crossref PubMed Scopus (63) Google Scholar, Takei et al., 1995Takei K. McPherson P.S. Schmid S.L. De Camilli P. Tubular membrane invaginations coated by dynamin rings are induced by GTP-γ S in nerve terminals.Nature. 1995; 374: 186-190Crossref PubMed Scopus (635) Google Scholar). Furthermore, in the presence of negatively charged lipid, dynamin self-assembles into helical tubes that constrict upon addition of GTP (Danino et al., 2004Danino D. Moon K.H. Hinshaw J.E. Rapid constriction of lipid bilayers by the mechanochemical enzyme dynamin.J. Struct. Biol. 2004; 147: 259-267Crossref PubMed Scopus (129) Google Scholar, Sweitzer and Hinshaw, 1998Sweitzer S.M. Hinshaw J.E. Dynamin undergoes a GTP-dependent conformational change causing vesiculation.Cell. 1998; 93: 1021-1029Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). This conformational change is believed to mimic the constriction at the necks of coated pits during endocytosis—a constriction that may lead to membrane fission contingent on the environment of the pit (Roux et al., 2006Roux A. Uyhazi K. Frost A. De Camilli P. GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission.Nature. 2006; 441: 528-531Crossref PubMed Scopus (370) Google Scholar). The GTPase domain of dynamin is conserved among all dynamin family members, and residues within G domains (Figure 1B; yellow boxes), which form the nucleotide-binding site, are conserved between dynamin and other GTPases including Ras. Mutational analyses have targeted conserved residues known to be functionally significant based on homology to other GTPases. Specifically, mutating K44, S45, and T65 causes a negative effect on dynamin function due to defects in GTP binding and/or hydrolysis (Damke et al., 2001Damke H. Binns D.D. Ueda H. Schmid S.L. Baba T. Dynamin GTPase domain mutants block endocytic vesicle formation at morphologically distinct stages.Mol. Biol. Cell. 2001; 12: 2578-2589Crossref PubMed Scopus (146) Google Scholar, Herskovits et al., 1993Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. Effects of mutant rat dynamin on endocytosis.J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (388) Google Scholar, Marks et al., 2001Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. GTPase activity of dynamin and resulting conformation change are essential for endocytosis.Nature. 2001; 410: 231-235Crossref PubMed Scopus (353) Google Scholar, Song et al., 2004aSong B.D. Leonard M. Schmid S.L. Dynamin GTPase domain mutants that differentially affect GTP binding, GTP hydrolysis, and clathrin-mediated endocytosis.J. Biol. Chem. 2004; 279: 40431-40436Crossref PubMed Scopus (71) Google Scholar, van der Bliek et al., 1993van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.M. Schmid S.L. Mutations in human dynamin block an intermediate stage in coated vesicle formation.J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (578) Google Scholar). Temperature-sensitive (ts) mutants have also been localized to the GTPase domain of a homologous dynamin gene product, shibire, in Drosophila (Chen et al., 1991Chen M.S. Obar R.A. Schroeder C.C. Austin T.W. Poodry C.A. Wadsworth S.C. Vallee R.B. Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis.Nature. 1991; 351: 583-586Crossref PubMed Scopus (424) Google Scholar, van der Bliek and Meyerowitz, 1991van der Bliek A.M. Meyerowitz E.M. Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic.Nature. 1991; 351: 411-414Crossref PubMed Scopus (574) Google Scholar). At nonpermissive temperatures, shibire ts mutants are defective in vesicle release, and a dynamin collar is observed at the necks of accumulating coated pits (Koenig and Ikeda, 1989Koenig J.H. Ikeda K. Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval.J. Neurosci. 1989; 9: 3844-3860Crossref PubMed Google Scholar). Equivalent mutations in humans also exhibit a defect in endocytosis and GTPase activity at nonpermissive temperatures (Damke et al., 1995Damke H. Baba T. van der Bliek A.M. Schmid S.L. Clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin.J. Cell Biol. 1995; 131: 69-80Crossref PubMed Scopus (335) Google Scholar, Narayanan et al., 2005Narayanan R. Leonard M. Song B.D. Schmid S.L. Ramaswami M. An internal GAP domain negatively regulates presynaptic dynamin in vivo: a two-step model for dynamin function.J. Cell Biol. 2005; 169: 117-126Crossref PubMed Scopus (52) Google Scholar). In addition to mutation studies, X-ray structures of GTPase domains from Dictyostelium discoideum dynamin A (dyn A) (Niemann et al., 2001Niemann H.H. Knetsch M.L. Scherer A. Manstein D.J. Kull F.J. Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms.EMBO J. 2001; 20: 5813-5821Crossref PubMed Scopus (92) Google Scholar) and Rattus norvegicus dynamin 1 (Reubold et al., 2005Reubold T.F. Eschenburg S. Becker A. Leonard M. Schmid S.L. Vallee R.B. Kull F.J. Manstein D.J. Crystal structure of the GTPase domain of rat dynamin 1.Proc. Natl. Acad. Sci. USA. 2005; 102: 13093-13098Crossref PubMed Scopus (54) Google Scholar) demonstrate that the core architecture of the dynamin GTPase domain is conserved when compared with other GTPase structures. Along with the GTPase domain, dynamin contains a middle domain, a pleckstrin homology (PH) domain, a GTPase effector domain (GED), and a C-terminal proline-rich domain (PRD) (Figures 1A and 1B). The middle domain and GED promote self-assembly, whereas the PH domain and PRD target dynamin to sites of vesicle scission. The PRD has been shown to interact with Src homology 3 (SH3) domains in proteins involved in endocytosis, which recruit dynamin to sites of action and may modulate dynamin activity (Schmid et al., 1998Schmid S.L. McNiven M.A. De Camilli P. Dynamin and its partners: a progress report.Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (351) Google Scholar). Most dynamin-related proteins lack the PH and PRD domains, so the roles of these domains are specific for dynamin. The PH domain is essential for interactions with lipid bilayers (Salim et al., 1996Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I. Driscoll P.C. et al.Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase.EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (481) Google Scholar). Based on X-ray structures (Ferguson et al., 1994Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Crystal structure at 2.2 Å resolution of the pleckstrin homology domain from human dynamin.Cell. 1994; 79: 199-209Abstract Full Text PDF PubMed Scopus (238) Google Scholar, Timm et al., 1994Timm D. Salim K. Gout I. Guruprasad L. Waterfield M. Blundell T. Crystal structure of the pleckstrin homology domain from dynamin.Nat. Struct. Biol. 1994; 1: 782-788Crossref PubMed Scopus (104) Google Scholar), the core folds of the dynamin PH domain are conserved when compared to PH domains from other proteins. Complementary NMR studies of the dynamin PH domain reveal dynamic motions in variable loops (Figure 1B, red boxes) that interact with lipid head groups (Fushman et al., 1995Fushman D. Cahill S. Lemmon M.A. Schlessinger J. Cowburn D. Solution structure of pleckstrin homology domain of dynamin by heteronuclear NMR spectroscopy.Proc. Natl. Acad. Sci. USA. 1995; 92: 816-820Crossref PubMed Scopus (82) Google Scholar). During self-assembly of dynamin onto lipid surfaces, the variable loops preferentially bind negatively charged bilayers (Zheng et al., 1996Zheng J. Cahill S.M. Lemmon M.A. Fushman D. Schlessinger J. Cowburn D. Identification of the binding site for acidic phospholipids on the pH domain of dynamin: implications for stimulation of GTPase activity.J. Mol. Biol. 1996; 255: 14-21Crossref PubMed Scopus (212) Google Scholar), which stimulates GTP hydrolysis (Tuma et al., 1993Tuma P.L. Stachniak M.C. Collins C.A. Activation of dynamin GTPase by acidic phospholipids and endogenous rat brain vesicles.J. Biol. Chem. 1993; 268: 17240-17246Abstract Full Text PDF PubMed Google Scholar). Mutations in these loops result in a decrease in lipid binding, assembly-stimulated GTPase activity, and endocytosis (Lee et al., 1999Lee A. Frank D.W. Marks M.S. Lemmon M.A. Dominant-negative inhibition of receptor-mediated endocytosis by a dynamin-1 mutant with a defective pleckstrin homology domain.Curr. Biol. 1999; 9: 261-264Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, Vallis et al., 1999Vallis Y. Wigge P. Marks B. Evans P.R. McMahon H.T. Importance of the pleckstrin homology domain of dynamin in clathrin-mediated endocytosis.Curr. Biol. 1999; 9: 257-260Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), demonstrating that dynamin-lipid interactions are essential. Furthermore, mutations in the PH domain of dynamin 2, the ubiquitously expressed isoform, lead to a human peripheral neuropathy, Charcot-Marie-Tooth disease (Zuchner et al., 2005Zuchner S. Noureddine M. Kennerson M. Verhoeven K. Claeys K. De Jonghe P. Merory J. Oliveira S.A. Speer M.C. Stenger J.E. et al.Mutations in the pleckstrin homology domain of dynamin 2 cause dominant intermediate Charcot-Marie-Tooth disease.Nat. Genet. 2005; 37: 289-294Crossref PubMed Scopus (273) Google Scholar). The middle domain of dynamin not only links the GTPase and PH domains but also plays a role in regulating self-assembly (Smirnova et al., 1999Smirnova E. Shurland D.L. Newman-Smith E.D. Pishvaee B. van der Bliek A.M. A model for dynamin self-assembly based on binding between three different protein domains.J. Biol. Chem. 1999; 274: 14942-14947Crossref PubMed Scopus (113) Google Scholar). In vitro mutation studies of dynamin (Ramachandran et al., 2007Ramachandran R. Surka M. Chappie J.S. Fowler D.M. Foss T.R. Song B.D. Schmid S.L. The dynamin middle domain is critical for tetramerization and higher-order self-assembly.EMBO J. 2007; 26: 559-566Crossref PubMed Scopus (132) Google Scholar) and a dynamin-related protein in yeast (DNM1) (Ingerman et al., 2005Ingerman E. Perkins E.M. Marino M. Mears J.A. McCaffery J.M. Hinshaw J.E. Nunnari J. Dnm1 forms spirals that are structurally tailored to fit mitochondria.J. Cell Biol. 2005; 170: 1021-1027Crossref PubMed Scopus (410) Google Scholar) show that the middle domain is required for efficient self-assembly into higher-ordered structures. In addition, missense mutations in the middle domain of dynamin 2 are linked to another human disease, autosomal dominant centronuclear myopathy (Bitoun et al., 2005Bitoun M. Maugenre S. Jeannet P.Y. Lacene E. Ferrer X. Laforet P. Martin J.J. Laporte J. Lochmuller H. Beggs A.H. et al.Mutations in dynamin 2 cause dominant centronuclear myopathy.Nat. Genet. 2005; 37: 1207-1209Crossref PubMed Scopus (309) Google Scholar). Unlike other GTPases that require a GTPase-activating protein (GAP), dynamin's GED promotes self-assembly and stimulates its own GTPase activity (Song et al., 2004bSong B.D. Yarar D. Schmid S.L. An assembly-incompetent mutant establishes a requirement for dynamin self-assembly in clathrin-mediated endocytosis in vivo.Mol. Biol. Cell. 2004; 15: 2243-2252Crossref PubMed Scopus (62) Google Scholar). Addition of isolated GED to unassembled dynamin stimulates GTPase activity in a highly cooperative manner (Sever et al., 2000Sever S. Damke H. Schmid S.L. Dynamin:GTP controls the formation of constricted coated pits, the rate limiting step in clathrin-mediated endocytosis.J. Cell Biol. 2000; 150: 1137-1148Crossref PubMed Scopus (190) Google Scholar), and suppressor mutants of a temperature-sensitive mutation in the GTPase domain (shibire ts2) were identified in the GED, confirming its role in regulating GTPase activity (Narayanan et al., 2005Narayanan R. Leonard M. Song B.D. Schmid S.L. Ramaswami M. An internal GAP domain negatively regulates presynaptic dynamin in vivo: a two-step model for dynamin function.J. Cell Biol. 2005; 169: 117-126Crossref PubMed Scopus (52) Google Scholar). However, in addition to the self-regulatory properties of dynamin, phospholipase D has been proposed to act as an external GAP for dynamin and accelerates endocytosis of epidermal growth factor receptor (Lee et al., 2006Lee C.S. Kim I.S. Park J.B. Lee M.N. Lee H.Y. Suh P.G. Ryu S.H. The phox homology domain of phospholipase D activates dynamin GTPase activity and accelerates EGFR endocytosis.Nat. Cell Biol. 2006; 8: 477-484Crossref PubMed Scopus (103) Google Scholar). Cryo-electron microscopy (cryo-EM) has been useful for elucidating structural features of dynamin 1, the neuronal-specific isoform (Zhang and Hinshaw, 2001Zhang P. Hinshaw J.E. Three-dimensional reconstruction of dynamin in the constricted state.Nat. Cell Biol. 2001; 3: 922-926Crossref PubMed Scopus (197) Google Scholar). Three-dimensional density maps of a dynamin mutant, lacking its C-terminal proline-rich domain (ΔPRD), in the constricted and nonconstricted states reveal a T-shaped subunit consisting of three prominent radial densities: inner, middle, and outer (Figure 1C) (Chen et al., 2004Chen Y.J. Zhang P. Egelman E.H. Hinshaw J.E. The stalk region of dynamin drives the constriction of dynamin tubes.Nat. Struct. Mol. Biol. 2004; 11: 574-575Crossref PubMed Scopus (120) Google Scholar, Zhang and Hinshaw, 2001Zhang P. Hinshaw J.E. Three-dimensional reconstruction of dynamin in the constricted state.Nat. Cell Biol. 2001; 3: 922-926Crossref PubMed Scopus (197) Google Scholar). Manual docking of crystal structures for the GTPase domain from human guanylate-binding protein (a distantly related dynamin family member) and the PH domain from human dynamin 1 to the constricted ΔPRD dynamin tube suggested that these domains reside in the outer and inner radial densities, respectively (Zhang and Hinshaw, 2001Zhang P. Hinshaw J.E. Three-dimensional reconstruction of dynamin in the constricted state.Nat. Cell Biol. 2001; 3: 922-926Crossref PubMed Scopus (197) Google Scholar). In this study, we predict conformational changes that occur during constriction using real-space refinement methods. This automated technique places crystal structures of the mammalian GTPase and PH domains from dynamin in both the constricted and nonconstricted states of ΔPRD dynamin. We define conformational motions of the crystal structures in the outer and inner radial densities that lead to the observed constriction. Furthermore, proteolysis and immunogold labeling studies verify the positions of dynamin structural domains observed in our fittings. From these results, we can predict interactions between and motions in the middle domain and GED based on topologic restraints. Overall, our fittings provide a model where repeating subunits in the ΔPRD dynamin helical array undergo a corkscrew motion during constriction consistent with conformational changes observed experimentally. Dynamin readily forms ordered tubes in the presence of negatively charged liposomes. Both full-length and ΔPRD dynamin tubes constrict and twist upon GTP hydrolysis; however, only ΔPRD dynamin constricts in the presence of nonhydrolyzable GTP analogs. Three-dimensional reconstructions of ΔPRD dynamin tubes in the constricted and nonconstricted states reveal the conformational change that occurs upon GTP binding (Chen et al., 2004Chen Y.J. Zhang P. Egelman E.H. Hinshaw J.E. The stalk region of dynamin drives the constriction of dynamin tubes.Nat. Struct. Mol. Biol. 2004; 11: 574-575Crossref PubMed Scopus (120) Google Scholar). Specifically, the middle radial density (Figure 1C) undergoes a dramatic rearrangement such that this region becomes highly kinked in the constricted state (compare red lines in Figures 2C and 2D). The outer and inner radial densities (Figure 1C) also rearrange during constriction, and until now it remained unclear what these conformational changes represent at the molecular level. To address this question, the atomic structures of the rat dynamin GTPase (99% identical to human; Reubold et al., 2005Reubold T.F. Eschenburg S. Becker A. Leonard M. Schmid S.L. Vallee R.B. Kull F.J. Manstein D.J. Crystal structure of the GTPase domain of rat dynamin 1.Proc. Natl. Acad. Sci. USA. 2005; 102: 13093-13098Crossref PubMed Scopus (54) Google Scholar) and human dynamin 1 PH (Timm et al., 1994Timm D. Salim K. Gout I. Guruprasad L. Waterfield M. Blundell T. Crystal structure of the pleckstrin homology domain from dynamin.Nat. Struct. Biol. 1994; 1: 782-788Crossref PubMed Scopus (104) Google Scholar) domains were fit to the cryo-EM reconstructions of human dynamin 1 (Chen et al., 2004Chen Y.J. Zhang P. Egelman E.H. Hinshaw J.E. The stalk region of dynamin drives the constriction of dynamin tubes.Nat. Struct. Mol. Biol. 2004; 11: 574-575Crossref PubMed Scopus (120) Google Scholar) with a molecular modeling program, Yammp (Tan and Harvey, 1993Tan R.K.-Z. Harvey S.C. Yammp: development of a molecular mechanics program using the modular programming method.J. Comput. Chem. 1993; 14: 455-470Crossref Scopus (37) Google Scholar), using rigid body Monte Carlo with simulated annealing. The crystal structures are fit to the cryo-EM maps using the vector lattice (VLAT) component of Yammp, which defines the electron density as a 3D potential. To sample possible orientations, a reduced-representation (Cα atoms) GTPase domain was iteratively moved at random as a rigid unit using Monte Carlo with simulated annealing. The score of the fitting, based on the VLAT term, improves until an orientation that best matches the experimental data is found. The GTPase domain was initially placed at an arbitrary location relative to the cryo-EM map. After several iterations of rigid body refinement, the structure was determined to have the best fit in the outer radial density of the cryo-EM maps. A reasonable fit was not found in the density near the lipid bilayer or in the middle radial density. The reconstruction of ΔPRD dynamin tubes in the nucleotide-bound (constricted) state identified 13.2 subunits per turn of the helix (Chen et al., 2004Chen Y.J. Zhang P. Egelman E.H. Hinshaw J.E. The stalk region of dynamin drives the constriction of dynamin tubes.Nat. Struct. Mol. Biol. 2004; 11: 574-575Crossref PubMed Scopus (120) Google Scholar). Scanning transmission electron microscopy (STEM) analysis of dynamin tubes revealed ∼30 dynamin molecules per turn (Zhang and Hinshaw, 2001Zhang P. Hinshaw J.E. Three-dimensional reconstruction of dynamin in the constricted state.Nat. Cell Biol. 2001; 3: 922-926Crossref PubMed Scopus (197) Google Scholar), suggesting each repeating subunit contains a dimer. This dimer is evident when the density is viewed at higher thresholds (yellow density boxed in Figures 2C and 2D) and appears to be asymmetric. Two GTPase monomers were initially fit to the asymmetric repeat in the density (one of the 13 equivalent subunits) using rigid body Monte Carlo with simulated annealing. The best fit was determined for each monomer in the dimer pair, and 13 dimers (26 GTPase monomers) with this configuration were then fit to a complete turn of the constricted ΔPRD dynamin helix (Figure 2, green ribbons). The final model provides the optimal fit of dynamin GTPase domains to the helical density in the constricted state (Figures 2B and 2D). We used a similar method to define the best fit of the GTPase domain to the nonconstricted helical density, which contains 14.2 repeating subunits per turn corresponding to ∼28 dynamin monomers. Briefly, we again fit two monomers to a repeating subunit of the helix using rigid body refinement. To prevent bias, GTPase monomers were placed at initial orientations distinct from the dimer orientation determined for the constricted state. The best orientation for the monomers in the repeating subunit was determined after several rounds of refinement, and from this model, 14 dimer subunit orientations were then fit to one turn of the nonconstricted helix (Figures 2A and 2C; 28 GTPase monomers). In both fittings, the final placements for the GTPase domain position the N- and C-terminal helices toward the cleft of the dynamin structure (Figure 3, light and dark purple helices), where they form a hydrophobic cleft that has been proposed to bind the GED (Niemann et al., 2001Niemann H.H. Knetsch M.L. Scherer A. Manstein D.J. Kull F.J. Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms.EMBO J. 2001; 20: 5813-5821Crossref PubMed Scopus (92) Google Scholar). In addition, the shibire ts1 mutation (G273D) resides immediately upstream of the C-terminal helix of the GTPase domain (Figure 3, red spheres). The C-terminal helix is also in an orientation where contiguous sequence would continue toward the middle radial density. This is consistent with the placement of the middle domain within the middle radial density. For comparison, a nearly identical placement of the GTPase domain was observed when the crystal structure from Dictyostelium dyn A (60% identical to human; Niemann et al., 2001Niemann H.H. Knetsch M.L. Scherer A. Manstein D.J. Kull F.J. Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms.EMBO J. 2001; 20: 5813-5821Crossref PubMed Scopus (92) Google Scholar) was fit to the density (Figure 3, insert). Dyn A, which is involved in mitochondrial division, has additional sequence between the G2 and G3 regions of the GTPase when compared with rat and human dynamin. Not surprisingly, this additional loop (circled in red) cannot be accommodated when fit to the ΔPRD dynamin structure and protrudes away from the density. The proximity of GTPase monomers due to helical packing is close enough to allow intermolecular interactions. Specifically, adjacent dynamin GTPases along the ridge of outer radial density are in a position to interact with one another, while connections across the cleft of the helical array are likely maintained via middle-GED interactions (Figures 4A and 4B). Based on the fittings of the rat GTPase crystal structure to the constricted and nonconstricted densities, the interface within the GTPase dimer remains largely unchanged (Figure 4C, interface 1). This interface is comprised of a highly conserved sequence near the switch 2 region (Figures 4A and 4B, gold ribbon), and the shibire ts2 mutation (G146, colored orange in Figure 4C) resides near this interface (Narayanan et al., 2005Narayanan R. Leonard M. Song B.D. Schmid S.L. Ramaswami M. An internal GAP domain negatively regulates presynaptic dynamin in vivo: a two-step model for dynamin function.J. Cell Biol. 2005; 169: 117-126Crossref PubMed Scopus (52) Google Scholar). Whereas the dimer interface is preserved after constriction, differences between adjacent GTPase dimers are apparent in the outer radial density (Figure 4C, interface 2). In the nonconstricted state, GTPase dimers are further away from each other than in the constricted state (Figure 4C, green versus blue ribbons). Upon nucleotide binding, the dimers undergo a subtle rotation (∼10°) that allows the subunits to pack more closely when constricted. Notably, the sequence at interface 2 is unique to dynamin family members (Figures 4A and 4B, blue and red ribbon, and also underlined in Figure 1B), and the amino acids in this region are more charged when compared with the rest of the GTPase sequence. The shibire ts1 mutation G273D (Figure 4C, Cα colored red) is found adjacent to this interface and has been proposed to uncouple the GTPase domain from the C-terminal half of dynamin (Damke et al., 1995Damke H. Baba T. van der Bliek A.M. Schmid S.L. Clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin.J. Cell Biol. 1995; 131: 69-80Crossref PubMed Scopus (335) Google Scholar). The position of this conserved glycine is proximal to the proposed GED-binding site (the aforementioned hydrophobic cleft), and therefore may act as a hinge between two sites where GTPase-GTPase and GTPase-GED interactions may sense nucleotide binding and drive helical constriction, respectively. The human dynamin 1 PH domain crystal structure (Ferguson et al., 1994Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Crystal structure at 2.2 Å resolution of the pleckstrin homology domain from human dynamin.Cell. 1994; 79: 199-209Abstract Full Text PDF PubMed Scopus (238) Google Scholar) was fit to the cryo-EM density in both the nonconstricted and constricted states using rigid body Monte Carlo with simulated annealing (Figures 2E and 2F). In the nonconstricted map, the molecular fitting places the PH domain in the inner radial density at the lipid bilayer interface. The density in this region is well defined with a contour that matches the shape of the X-ray structure, where the density is more tapered near the middle radial density and is wider near the lipid interface. Therefore, the variable loops are positioned near the lipid bilayer with the N- and C-terminal ends adjacent to the middle radial density (Figure 3A). This N-terminal orientation is consistent with placing the middle domain in the middle radial density between the GTPase and PH domain structures. The C-terminal sequence of the PH domain likely continues into the middle radial density as well, allowing the GED to interact with the middle and GTPase domains as predicted previously (Narayanan et al., 2005Narayanan" @default.
• W2058359969 created "2016-06-24" @default.
• W2058359969 creator A5008494368 @default.
• W2058359969 creator A5033955219 @default.
• W2058359969 creator A5068703722 @default.
• W2058359969 date "2007-10-01" @default.
• W2058359969 modified "2023-10-12" @default.
• W2058359969 title "A Corkscrew Model for Dynamin Constriction" @default.
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