title: Identification and function of conformational dynamics in the multidomain GTPase dynamin.
abstract: 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 α2(S) 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.
authors.affiliation: Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX, USA.
authors.forename: Saipraveen
authors.initials: S
authors.lastname: Srinivasan
authors.affiliation: Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL, USA.
authors.forename: Venkatasubramanian
authors.initials: V
authors.lastname: Dharmarajan
authors.affiliation: Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX, USA.
authors.forename: Dana Kim
authors.initials: DK
authors.lastname: Reed
authors.affiliation: Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL, USA.
authors.forename: Patrick R
authors.initials: PR
authors.lastname: Griffin
authors.affiliation: Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX, USA sandra.schmid@utsouthwestern.edu.
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Schmid
mesh_terms: D002460:Cell Line; D034281:Dynamins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D017384:Sequence Deletion
chemical_list: D050505:Mutant Proteins; D006160:Guanosine Triphosphate; D034281:Dynamins
keywords: centronuclear myopathy; clathrin‐mediated endocytosis; hydrogen–deuterium exchange; membrane fission; pleckstrin homology domain