Building a fission machine--structural insights into dynamin assembly and activation

Abstract

Dynamin is a large multidomain GTPase that assembles into helical arrays around the necks of deeply invaginated clathrin-coated pits and catalyzes membrane fission during the final stages of endocytosis. Although it is well established that the function of dynamin in vivo depends on its oligomerization and its capacity for efficient GTP hydrolysis, the molecular mechanisms governing these activities have remained poorly defined. In recent years, there has been an explosion of structural data that has provided new insights into the architecture, organization and nucleotide-dependent conformational changes of the dynamin fission machine. Here, we review the key findings of these efforts and discuss the implications of each with regard to GTP hydrolysis, dynamin assembly and membrane fission.

Keywords: Dynamin; Endocytosis; GTPase; Hydrolysis; Membrane fission; Structure.

Fig. 1.

Domain architecture of dynamin family members. Representative members of different dynamin-related proteins are shown. Hs, Homo sapiens; Sc, Saccharomyces cerevisiae; At, Arabidopsis thaliana; BSE, bundle-signaling element; PH, pleckstrin homology domain; GED, GTPase effector domain; PRD, proline- and arginine-rich domain; MTS, mitochondrial-targeting sequence; TM, transmembrane domain; EH, epsin homology domain.

Fig. 2.

Structures and crystal packing of dynamin family members. (A) X-ray structures of intact human dynamin 1, rat dynamin and human MxA. Each protein contains different mutations that yield stable dimers in solution. Only the monomers are shown. The linear domain arrangement of human dynamin 1 is shown above for comparison. Coloring is as follows: G domain, purple; BSE, yellow; middle–GED stalk, green; PH domain, blue. (B) Dynamin-related proteins crystallize in linear arrays that are mediated primarily by stalk–stalk interactions. Rat dynamin crystal packing (PDB: 3ZVR) is depicted in two orientations (top view above, side view below). (C) Magnified view of the dynamin stalk illustrating the three interfaces (circles) that mediate intermolecular interactions within the crystallized linear dynmain array. Mutations that result in the monodisperse dimers used for crystallization map to interface 3. (D) Disordered regions at the base of the stalk yield an ambiguity (black arrows) in determining the stalk–PH connectivity. (E) Different putative domain connectivities within the mutant dynamin dimers can conform to the same crystal packing. Alternate dimers are colored orange and light blue. The light gray surface illustrates how different arrangements all yield the same overall shape and packing.

Fig. 3.

Dynamin assembly and subunit architectures. (A) Pseudo-atomic model of the assembled dynamin polymer (PDB: 3ZYS) that has been derived from computationally fitting GG_GMPPCP (purple and yellow; PDB: 3ZYC), the MxA stalk (green, PDB: 3LJB) and the human dynamin 1 PH domain (blue, PDB: 1DYN) into the 12.2 Å GMPPCP-stabilized ΔPRD cryo-EM map (gray, EMD-1949). End-on and side-on views are shown in the left and right panels, respectively, with the dimensions and helical axis marked. (B) Cross-section view of the assembled dynamin polymer oriented as in A. The G domain, middle–GED stalk and PH domain occupy the head, stalk and leg density regions, respectively. The inner luminal diameter is indicated. M, membrane bilayer. (C) Different model representations of the minimal dynamin dimer building blocks. Monomers are colored purple and cyan. Left, dimer based on crystal packing (PDB: 3ZVR) that is stabilized by interface 2 interactions; center, X-shaped short dimer based on chemical crosslinking and computational docking that is stabilized by a domain swap of the C_GED helix; right, M-shaped long dimer based on chemical crosslinking and computational docking that is stabilized by a full domain swap of the GED. (D) Putative structure of membrane-bound dynamin tetramer. Underlying dimers are colored yellow and light blue. This model assumes the entire GED is domain-swapped in each monomer (see text). In this context, portions of interface 2 and 3 mediate inter-dimer interactions (gray box). The structure of the crystal packing dimer is shown on the right for comparison with each monomer colored yellow and light blue. Note that in this case, interface 2 and 3 form intra-dimer interactions (gray box).

Fig. 4.

The catalytic machinery of dynamin. (A) Domain arrangement of the G-domain–GED fusion derived from human dynamin 1. Each monomer contains a G domain core (purple), three helical segments (N_GTPase, C_GTPase and C_GED; yellow) that together form the bundle signaling element (BSE), and a flexible linker (dashed line). (B) Structure of the transition-state-stabilized dimer (PDB: 2X2E). The monomer cores are colored purple and cyan, and the bundle signaling elements (BSEs) are in yellow. (C) Switch region I (red) and switch region II (green) are stabilized at the dimer interface in the GG transition-state complex. GG monomers are shown in cyan and orange. The structure of the Giα₁–RGS4 transition-state complex is shown below for comparison (Giα₁ in cyan, RGS4 GAP in orange). Note that in this case the switch regions are stabilized at the interface between the G-protein and the GAP. (D) Transition-state stabilization of the of the GG active site. Structural elements involved in this stabilization are labeled. Catalytic and bridging waters are depicted as red spheres; bound Mg²⁺ and charge-compensating Na⁺ ions are shown as green and blue spheres, respectively. Dashed lines indicate hydrogen-bonding interactions. Together, the charge compensating cation, the bound Mg²⁺ and the K44 side-chain act to neutralize the negative charge that develops when the bond is broken between the β- and γ-phosphates during hydrolysis. (E) Conformational changes in the active site that accompany the formation of a G domain dimer in dynamin. monomer colored purple; nucleotide free rat dynamin G domain structure (PDB: 2AKA) colored green. These structural changes serve to optimally position the catalytic machinery, which in turn promotes stimulated GTP hydrolysis.

Fig. 5.

Structural constraints of G domain dimerzation. (A) Membrane-bound conformation of the dynamin tetramer (magenta). The dashed black line illustrates the axis of radial assembly within a helical rung. The arrows depict the relative orientation of the G domains within the tetramer. Note that the G domains do not form productive head-to-head interactions required for G domain dimerization. (B) Pseudoatomic model of the dynamin polymer generated by computationally docking crystallized dynamin domain structures into the GMPPCP-stabilized ΔPRD cryo-EM map (gray). The numbering and rainbow coloring (magenta to orange) denotes the sequential addition of tetramers to the assembly and terminates when the first G domain dimer is formed between magenta tetramer 1 and orange tetramer 5. The upper panel depicts a side view perpendicular to the helical axis; the lower panel is an end view looking down this axis. The sequential rungs of the dynamin helix are marked in the upper panel with black brackets. The areas indicated by the black boxes (see C and D) highlight that G domain dimerization only occurs between tetramers in adjacent helical rungs. (C,D) Magnified top (C) and side (D) views of G domain interactions within the assembled dynamin polymer. The crystallized GG_GMPPCP dimer (monomers colored magenta and orange) is shown below each for comparison. (E) Illustration of G domain dimerization in the context of the helical polymer. Tetramers are colored and numbered as in B. Arrows denote the direction of the hydrolysis-dependent BSE conformational change that would accompany stimulated GTP hydrolysis in each G domain dimer pair.

Fig. 6.

The dynamin powerstroke and conformational coupling. (A) Structural superposition of GG_GMPPCP (purple, PDB: 3ZYC) and (cyan, PDB: 2X2E) highlighting alternative BSE conformations. The BSE hinge is colored blue. (B) Side view of the hydrolysis-dependent BSE conformational change that constitutes the dynamin powerstroke. GG_GMPPCP and monomers are colored as in A and the BSE helices are labeled. The red arrow labeled ‘powerstroke’ depicts the 69° downwards rotation of the BSE in the transition-state complex. (C,D) Conformational coupling of the β-sheet, switch I and BSE movements during GTP loading (C) and GTP hydrolysis (D). GG_GMPPCP (purple, PDB: 3ZYC), (cyan, PDB: 2X2E) and the G domain from the nucleotide-free, full-length rat dynamin crystal structure (yellow, PDB: 3ZVR) are superimposed. Arrows indicate the conformational changes between the different structures.