Prof. Dr. Oliver Daumke, MDC
Prof. Dr. Christian Spahn, Charité
This project deals with the structural and functional examination of proteins forming GTP- and ATP-dependent membrane scaffolds. We previously showed that the septin-related GTPase of Immunity Associated protein 2 (GIMAP2) establishes a GTP-dependent protein scaffold at the surface of lipid droplets (Figure 1). This scaffold is intrinsically stable since GIMAP2 cannot hydrolyze GTP on its own. Using co-localization studies, we now discovered that GIMAP7 also localizes to lipid droplets. In contrast to GIMAP2, GIMAP7 efficiently hydrolyzes GTP in a concentration-dependent manner. We found that GIMAP7 dimerizes in response to GTP-binding and determined the structure of the GTP-bound GIMAP7 dimer. In combination with mutagenesis data, we demonstrated that GIMAP7 provides a catalytic arginine finger into the opposing GTPase site during dimerization. We also showed that GIMAP7 can stimulate the GTPase of GIMAP2 via an analogous mechanism. We therefore suggest that GIMAP7 acts as a regulator of GIMAP2 scaffold disassembly.
In the coming funding period, we will continue our efforts to study structure and function of GIMAP GTPases. To this end, we will explore the structural basis for the association of GIMAPs with their cellular interaction partners and determine the functional consequence of these interactions for the regulation of autophagy and apoptosis in lymphocytes. We will extend our structural studies to the dynamin-like Eps15 homology domain containing protein 2 (EHD2) ATPase, which forms a membrane-associated scaffold at the neck of caveolae. Based on our previous structural data of ATP-bound EHD2, we now aim to understand the structural changes associated with nucleotide binding and hydrolysis. To this end, we will determine structures of ADP- and nucleotide-free forms of EHD2. Cryo electron microscopy reconstructions will be employed to study the structure of membrane-bound EHD2 and the conformational changes associated with membrane binding and oligomerization. These studies will shed light on the function and mechanism of EHD2 at caveolae and on the general assembly mechanism of EHD proteins. Furthermore, they will allow us to compare the principles of assembly on membranes and the role of nucleotide hydrolysis in septin and dynamin superfamily proteins.
Fig. 1: GTP-dependent scaffold formation of GIMAP2.
(top) Structure of the monomeric nucleotide-free GIMAP2 shows a Ras-like G domain (in green) with two additional amphipathic helices a6 and a7 (in orange) folded against switch II (blue).
(bottom) In the absence of a7 and presence of GTP, GIMAP2 oligomerised via the G- and C-interfaces in the crystal to form a linear oligomer. In a full-length context, a7 might recruit interaction partners to this membrane-associated GIMAP scaffold. The conversion between the monomeric and oligomeric state is regulated by GTP binding or hydrolysis (red arrows) (from Schwefel et al., PNAS 2010, Schwefel et al., SGTP 2011).
We have also structurally characterized other membrane-associated protein scaffolds in this SFB. Examples include dynamin (in collaboration with Volker Haucke / A01 and Frank Noé / A04, dynamin-1-like protein or the stomatin protein (in collaboration with Kate Poole and Gary Lewin / A09).
Assoziierte Mitarbeiter: Dr. David Schwefel (until 2011), Janko Brand (until 2012), Chris Froehlich (until 2014), Arasu Balasubramaniyam, Alexej Dick
- Daumke O., Roux A., Haucke V. BAR domain-containing protein scaffolds in dynamin-mediated membrane fission. Cell 156: 882-892 (2014) (Review)
- Shah C., Hegde B.G., Morén B, Behrmann E., Mielke T., Moenke G., Spahn C.M.T., Lundmark R., Daumke O., Langen R. Structural insights into membrane interaction and caveolar targeting of dynamin-like EHD2. Structure 22: 409-20 (2014)
- Fröhlich C., Grabiger S., Schwefel D., Faelber K., Rosenbaum E., Mears J., Rocks O., Daumke O. Structural insights into oligomerization and mitochondrial remodelling of dynamin 1-like protein. EMBO J, doi: 10.1038/emboj.2013.74. (2013).
- Schwefel D., Arasu B.S., Marino S., Lamprecht B., Köchert K., Rosenbaum E.,Eichhorst J.., Wiesner B., Behlke J., Rocks O., Mathas S., Daumke O. Structural insights into the mechanism of GTPase activation in the GIMAP family. Structure 21, 550-9. (2013).
- Faelber K., Held M., Gao S., Posor Y., Haucke V., Noe F., and Daumke O. Structural insights into dynamin-mediated membrane fission. Structure 20, 1621-28 (2012) (Review).
- Brand, J., Smith, E.S., Schwefel, D., Lapatsina, L., Poole, K., Omerbašić, D., Kozlenkov, A., Behlke, J., Lewin, G.R., Daumke, O. A stomatin dimer modulates the activity of acid-sensing ion channels. EMBO J 31, 363546-46 (2012).
- Faelber K., Posor Y., Gao S., Held M., Roske Y., Schulze D., Haucke V., Noé F., Daumke O. Crystal structure of nucleotide-free dynamin. Nature 477, 556–560 (2011).
- Schwefel D., Daumke O. GTP-dependent scaffold formation in the GTPase of Immunity Associated Protein family. Small GTPases 2, 27-30 (2011) (Commentary)
- Schwefel D., Fröhlich C., Eichhorst J., Wiesner B., Behlke J., Aravind L., Daumke O. Structural basis of oligomerization in septin-like GTPase of immunity-associated protein2 (GIMAP2). Proc. Natl. Acad. Sci. 107, 20299-305 (2010).