Research Report 2010 - MDC

Structure of the GroupOliver DaumkeGroup LeaderDr. rer. nat.Oliver DaumkeScientistDr. Katja FälberGraduate StudentsDavid SchwefelSong GaoJanko BrandClaudio ShahChris FröhlichTechnical AssistantsSabine WernerMarion PapstSecretariatBirgit CloosStructure and Mechanism of MembraneremodelingG-proteinsGuanine nucleotide binding proteins (G-proteins) are involved in a diverse range of cellularprocesses including protein synthesis, sensual perception, vesicular transport and signaltransduction cascades. Whereas small G-proteins are molecular switches that cycle between anactive GTP-bound form and an inactive GDP-bound form, large G-proteins of the dynaminsuperfamily are mechano-chemical enzymes that use the energy of GTP hydrolysis to activelyremodel membranes. Members of both groups bind to membranes, and this interaction iscrucial for their function. Our projects aim to elucidate the interaction and reciprocalmodulation of membranes and G-proteins using structural, biochemical and cell-biologicalmethods.1. EHD as a molecular model for membraneremodelling G-ProteinsMembers of the dynamin superfamily are multidomainproteins with an amino-terminal G-domain. Itsfounding member dynamin oligomerises around theneck of clathrin-coated vesicles and induces vesicle scissionin a reaction which is dependent on GTP hydrolysis.The mechanism of vesicle scission is poorly understoodat the molecular level.In this project, we want to establish the less characterisedEHD family as a model system to understandprinciples of membrane remodelling in the dynaminsuperfamily. EHDs comprise a highly conserved eukaryoticprotein family with four members (EHD1-4) inmammals and a single member in C. elegans and D.melanogaster. The proteins are built of an amino-terminalG-domain, followed by a helical domain and a carboxy-terminalEH-domain known to interact with linearpeptide motifs of proteins involved in endocytosis.EHDs can be found at vesicular and tubular structuresin vivo, and EHD family members have been shown toregulate several trafficking pathways including the exitof cargo proteins from the endocytic recycling compartment.We could show that EHD2 binds with low affinity tonucleotides, like other members of the dynamin superfamily.Surprisingly, ATP rather than GTP was bound. Wedemonstrated that EHD2 could also bind to negativelycharged liposomes, and this binding resulted in thedeformation of the liposomes into long tubular structuresaround which EHD2 oligomerised in ring-likeoligomers (Figure 1a). Furthermore, the slow ATPaseactivity of EHD2 was enhanced in the presence of liposomes,which is another typical feature of dynaminrelatedproteins.We solved the crystal structure of an EHD2 dimer in thepresence of a non-hydrolysable ATP analogue (Figure1b). Dimerisation is mediated via a highly conserved surfacepatch in the G-domain. We could show that thelipid-binding sites in each dimer are located at the tip ofthe helical domains and create a highly curved lipidinteraction site, which might contribute to the membraneremodelling activity of EHD2. We further predictedthe architecture of the EHD2 oligomeric ring where20 EHD2 dimers assemble via the G-domain to form atightly packed oligomer (Figure 1c).Having solved the structure, we will continue this projectto understand the structural changes associated112 Cancer Research