Triphosphate induced dimerization of human guanylate binding protein 1 involves association of the C-terminal helices: a joint double electron-electron resonance and FRET study.

Human guanylate binding protein 1 (hGBP1) is a member of the dynamin superfamily of large GTPases. During GTP hydrolysis, the protein undergoes structural changes leading to self-assembly. Previous studies have suggested dimerization of the protein by means of its large GTPase (LG) domain and significant conformational changes in helical regions near the LG domain and at its C-terminus. We used site-directed labeling and a combination of pulsed electron paramagnetic resonance and time-resolved fluorescence spectroscopy for structural investigations on hGBP1 dimerization and conformational changes of its C-terminal helix α13. Consistent distance measurements by double electron-electron resonance (DEER, also named pulse double electron resonance = PELDOR) spectroscopy and Förster resonance energy transfer (FRET) measurements using model-free analysis approaches revealed a close interaction of the two α13 helices in the hGBP1 dimer formed upon binding of the nonhydrolyzable nucleoside triphosphate derivate GppNHp. In molecular dynamics (MD) simulations, these two helices form a stable dimer in solution. Our data show that dimer formation of hGBP1 involves multiple spatially distant regions of the protein, namely, the N-terminal LG domain and the C-terminal helices α13. The contacts formed between the two α13 helices and the resulting juxtaposition are expected to be a key step for the physiological membrane localization of hGBP1 through the farnesyl groups attached to the end of α13.