title: EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation.
abstract: The EH-domain-containing protein 2 (EHD2) is a dynamin-related ATPase that confines caveolae to the cell surface by restricting the scission and subsequent endocytosis of these membrane pits. For this, EHD2 is thought to first bind to the membrane, then to oligomerize, and finally to detach, in a stringently regulated mechanistic cycle. It is still unclear how ATP is used in this process and whether membrane binding is coupled to conformational changes in the protein. Here, we show that the regulatory N-terminal residues and the EH domain keep the EHD2 dimer in an autoinhibited conformation in solution. By significantly advancing the use of infrared reflection-absorption spectroscopy, we demonstrate that EHD2 adopts an open conformation by tilting the helical domains upon membrane binding. We show that ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is related to detachment of EHD2 from the membrane. Finally, we demonstrate that the regulation of EHD2 oligomerization in a membrane-bound state is crucial to restrict caveolae dynamics in cells.
authors.affiliation: Chemistry and Molecular Biology, Gothenburg University, 405 30 Gothenburg, Sweden.|Pharmaceutical Technology and Biopharmacy, University of Freiburg, 79104 Freiburg im Breisgau, Germany.|Centre for Biological Signalling Studies, Albert-Ludwigs-Universität, 79104 Freiburg im Breisgau, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Hoernke
authors.affiliation: Integrative Medical Biology, Umeå University, 901 87 Umea, Sweden.
authors.forename: Jagan
authors.initials: J
authors.lastname: Mohan
authors.affiliation: Medical Biochemistry and Biophysics, Laboratory for Molecular Infection Medicine Sweden, Umeå University, 901 87 Umea, Sweden.
authors.forename: Elin
authors.initials: E
authors.lastname: Larsson
authors.affiliation: Medical Biochemistry and Biophysics, Laboratory for Molecular Infection Medicine Sweden, Umeå University, 901 87 Umea, Sweden.
authors.forename: Jeanette
authors.initials: J
authors.lastname: Blomberg
authors.affiliation: Medical Biochemistry and Biophysics, Laboratory for Molecular Infection Medicine Sweden, Umeå University, 901 87 Umea, Sweden.
authors.forename: Dana
authors.initials: D
authors.lastname: Kahra
authors.affiliation: Chemistry and Molecular Biology, Gothenburg University, 405 30 Gothenburg, Sweden.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Westenhoff
authors.affiliation: Institute of Chemistry, Martin-Luther-Universität Halle-Wittenberg, 06099 Halle (Saale), Germany richard.lundmark@umu.se christian.schwieger@chemie.uni-halle.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Schwieger
authors.affiliation: Integrative Medical Biology, Umeå University, 901 87 Umea, Sweden; richard.lundmark@umu.se christian.schwieger@chemie.uni-halle.de.|Medical Biochemistry and Biophysics, Laboratory for Molecular Infection Medicine Sweden, Umeå University, 901 87 Umea, Sweden.
authors.forename: Richard
authors.initials: R
authors.lastname: Lundmark
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021941:Caveolae / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013055:Spectrophotometry, Infrared
chemical_list: D002352:Carrier Proteins; C485117:EHD2 protein, mouse; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate
keywords: EHD2; caveolae; infrared reflection–absorption spectroscopy; membrane-bound protein structure; membrane-reshaping protein