Vesicle release upon endocytosis requires membrane fission, catalyzed by the large GTPase dynamin. Dynamin contains five domains that together orchestrate its mechanochemical activity. Hydrogen–deuterium exchange coupled with mass spectrometry revealed global nucleotide‐ and membrane‐binding‐dependent conformational changes, as well as the existence of an allosteric relay element in the α2S helix of the dynamin stalk domain. As predicted from structural studies, FRET analyses detect large movements of the pleckstrin homology domain (PHD) from a ‘closed’ conformation docked near the stalk to an ‘open’ conformation able to interact with membranes. We engineered dynamin constructs locked in either the closed or open state by chemical cross‐linking or deletion mutagenesis and showed that PHD movements function as a conformational switch to regulate dynamin self‐assembly, membrane binding, and fission. This PHD conformational switch is impaired by a centronuclear myopathy‐causing disease mutation, S619L, highlighting the physiological significance of its role in regulating dynamin function. Together, these data provide new insight into coordinated conformational changes that regulate dynamin function and couple membrane binding, oligomerization, and GTPase activity during dynamin‐catalyzed membrane fission.
Dynamin‐catalyzed membrane fission requires long‐range nucleotide and/or membrane binding‐induced conformational changes and domain rearrangements that are identified and functionally characterized in this study.
Ligand‐induced conformational changes in dynamin were identified by HDX‐MS.
An allosteric relay helix, α2S, transmits conformational information from the G domain to the membrane and vice versa.
FRET analyses reveal conformational switches of the PH domain.
When locked in a closed conformation, the PH domain acts in an auto‐inhibitory fashion to regulate membrane binding and assembly.
The PH domain conformational switch is impaired in the centronuclear myopathy‐causing mutant Dyn2S619L.
The EMBO Journal (2016) 35: 443–457
- Received November 11, 2015.
- Revision received December 9, 2015.
- Accepted December 11, 2015.
- © 2016 The Authors
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