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62
Structural Insights into Dynamin-Catalyzed Membrane
Fission
Sandra Schmid 1 , Joshua Chappie 1, 2 , Fred Dyda 2 , Ya-Wen Liu 1 , Sylvia Neumann 1 , Thomas
Pucadyil 1 , Rajesh Ramachandran 1 .
The Scripps Research Institute 1 , National Institute of Diabetes and Digestive and Kidney
Diseases 2
Abstract:
Dynamin is a large, atypical GTPase that self-assembles to form rings and stacks of rings and
catalyzes membrane fission in the late stages of clathrin-mediated endocytosis. We have recently
reconstituted dynamin-catalyzed membrane fission from Supported Bilayers with Excess
Reservoir, SUPER templates, formed on silica beads and showed that fission occurs after cycles
of assembly and disassembly driven by GTP hydrolysis. Only GTPase restricted, self-limited
assemblies of dynamin can catalyze fission: long dynamin spirals pre-assembled in the absence
of GTP are inactive. Previous genetic and biochemical studies had implicated the C-terminus of
the GTPase effector domain (GED) in regulating dynamin’s GTPase activity, thus we engineered
a minimal GTPase-GED fusion protein (GG) that reconstitutes a robust GTPase activity
comparable to full-length dynamin and used x-ray crystallography to solve the structure of GG
in the presence of the transition-state analog GDP.AlF4- at 2.0Å resolution. This structure
reveals the first high-resolution view of dynamin’s GTPase domain in an activated conformation
and suggests a novel mechanism for dynamin stimulation that requires the dimerization of this
domain. The structure also provides key insights into the coupling of GTP hydrolysis with
dynamin disassembly and thus, into the mechanisms governing dynamin-catalyzed membrane
fission.