An Improved Model for Dynamin Assembly Revealed by Cryo-EM

Abstract

Dynamin is a multidomain GTPase that assembles into collar-like structures at the necks of deeply invaginated coated pits during the final stages of clathrin-mediated endocytosis (CME) and catalyzes membrane scission. Assembly of purified dynamin tetramers in vitro yields helical structures comparable to those observed in vivo. The formation of these oligomers stimulates dynamin's basal GTP hydrolysis >100-fold. Mutational analysis indicates that dynamin's stimulated GTP hydrolysis is required for CME; however, mounting evidence suggests that this activity causes disassembly of the dynamin collar rather than direct membrane severing. Despite recent structural studies showing that stimulated hydrolysis arises from the transition-dependent dimerization of dynamin's catalytic G domains, little is known about the conformational changes that precede and/or result from this interaction in the context of the polymer. Specifically, it is unclear how the G domains are properly oriented, which subunits associate, and how catalysis triggers dissociation of the pleckstrin homology (PH) domain at the membrane surface. Much of this ambiguity can be attributed to the low resolution (>20A) of previous dynamin polymer models and the absence of a complete dynamin tetramer crystal structure. To clarify these issues, we have used cryo-EM and iterative helical real space refinement to generate an 11A reconstruction of a truncated form of dynamin (ΔPRD) in the assembled, GMPPCP-bound state. This map reveals new structural characteristics including a twisted, interlacing interaction that stabilizes the middle/GED stalk and a previously uncharacterized density feature adjacent to the exterior GTPase head. Computational docking of crystallized dynamin fragments reveals the location and connectivity of different domains within the assembled polymer. Chemical crosslinking experiments also provide new insights into the architecture and organization of dynamin tetramer. These data have important implications regarding the conformational changes associated with dynamin catalyzed GTP hydrolysis and membrane fission.