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

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.

Fig. 1. Structure of GG dimer

a, Domain arrangement of GTPase-GED (GG) fusion constructed from human dynamin 1. Each monomer contains a GTPase core (green) and three helical segments: N_GTPase, C_GTPase (yellow) and C_GED (cyan). Dashed line denotes the flexible linker that tethers the C_GED helix. b, Structure of GDP.AlF₄⁻-stabilized GG dimer from the “long axis” crystal form shown from the side (left) and top (right). The GTPase cores of individual monomers are colored in green and blue. The helical segments in each monomer that constitute the bundle signaling element (BSE) are colored yellow with the C_GED helix highlighted in cyan. c, d, e, Structural interactions that stabilize the GG dimer. Coloring is the same as b. Key residues are labeled with a superscript (A or B) to indicate from which monomer they originate. The additional subscript (O or N) signifies the interaction of a main chain carbonyl or nitrogen. Dashed lines indicate hydrogen-bonding interactions.

Fig. 2. Catalytic machinery involved in dynamin GTP hydrolysis

a, Structure of GG active site. Red spheres labelled “C” and “B” denote catalytic and bridging waters respectively. Dashed lines indicate hydrogen-bonding interactions. b–d, Comparison of the charge-compensating elements in GG (b), hGBP1 (PDB: 2b92) (c), and MnmE (PDB: 2gj8) (d). Note the similar coordination by switch I in each case. e, K44 and the bound magnesium form similar charge-compensating interactions with the β-phosphate and aluminum fluoride. Portions of switch I and switch II have been removed for clarity. Red sphere labeled “C” denotes the catalytic water.

Fig. 3. Active site conformational changes induced by dynamin G-domain dimerization and GTP hydrolysis

a, A monomer from the GG dimer structure (green) is superimposed with the nucleotide free rat dynamin G domain structure (magenta, PDB: 2aka). The GDP.AlF₄⁻, magnesium, and active site waters from GG structure are shown. Coloring is the same in b, c, and g. Black arrows indicate structural movements induced by nucleotide binding and dimerization. b, P-loop tilting reorients Q40 and S41 to facilitate interactions with the bridging water and sodium ion respectively. c, GTP binding engages switch I, resulting in the coordination of the charge-compensating cation by the backbone carbonyl oxygens of G60 and G62. d–f, Conformational changes of the dynamin specific loop (colored blue) are mediated by a dense network of hydrogen bonding that includes switch I residues (red), as shown for d, nucleotide free rat dynamin GTPase domain (c, PDB: 2aka); e, GDP.AlF4--stabilized GG dimer (d, long axis crystal form), and f, GDP-bound Dictyostelium dynamin A (e, PDB: 1jwy). g, Switch II and the cis stabilizing loop undergo significant conformational rearrangements that precipitate hydrogen bonding between N112 and D147 and between the K113 carbonyl oxygen and Q148. These interactions stabilize switch II in cis, whereas interactions with the trans stabilizing loop across the dimer interface stabilize switch II in trans.

Fig. 4. Functional analyses of dynamin active site mutants

A, Q40, S41, and D180 are absolutely conserved among dynamin family members, but not more distantly related GTPases. Sequence alignment abbreviations are as follows: Hs, Homo sapiens; Rn, Rattus norvegicus; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae; Dd, Dictyostelium discoideum; Dyn, dynamin; Gm_Phragmo, Glycine max phragmoplastin; Hs_GBP1, interferon-γ-induced human guanylate binding protein; Np_BDLP, Nostoc punctiforme bacterial dynamin-like protein. b, Basal and c, assembly-stimulated GTPase activities of full-length wildtype and dynamin active site mutants (Q40E, S41A, and D180A) were determined as described in Methods. These data represent the average of at least three independent experiments with multiple independently purified batches of protein. d, Clathrin-mediated internalization of BSS-Tfn into MesNa-inaccessessible sealed vesicles and e, fluid-phase uptake of horse radish peroxidase into tTA-HeLa cells infected with adenoviruses expressing either WT or Q40E dynamin-1.

Fig. 5. Structure and conformational changes of the BSE

a, The BSE as viewed down the C_GED helix. Conserved hydrophobic side chains that comprise the GTPase-GED interface are shown in yellow. b, BSE conformational changes. Superposition shows a monomer from the GG dimer structure (green) superimposed with the nucleotide free rat dynamin G domain structure (PDB: 2aka, magenta). Black arrow indicates relative movement of BSE upon GTP hydrolysis. Asterisk denotes the position of conserved P294 that kinks the C_GTPase segment. A helix from the fused myosin motor domain mimics the C_GED segment in the rat dynamin structure. c, Relative orientation of the BSEs in the GG dimer. Structure is rotated 120° about the y-axis from the side view depicted in Fig. 1b. Dashed arrows indicate the assumed position of the middle/GED stalk in each monomer that is connected to the BSE in full-length dynamin. d, Cartoon illustrating C_GED’s association with the GTPase domain in GG and full-length dynamin. In GG, this interaction occurs in cis, producing a stable monomer that dimerizes only in the presence of a transition state mimic; in the full-length dynamin, C_GED interacts with the GTPase domain in trans, producing a stable dimer that associates further via middle/GED stalk interactions to form the tetramer. The transition state conformation of a membrane-bound minimal dynamin octamer as predicted from the GG dimer structure is also shown.