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 dirnerization 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 alpha 13. Consistent distance measurements by double electron electron resonance (DEER, also named pulse double electron resonance = PELDOR) spectroscopy and Forster resonance energy transfer (FRET) measurements using model-free analysis approaches revealed a close interaction of the two alpha 13 helices in the hGBP1 dinner 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 alpha 13. The contacts formed between the two alpha 13 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 alpha 13.