Integrative dynamic structural biology unveils conformers essential for the oligomerization of a large GTPase.

Guanylate binding proteins (GBPs) are soluble dynamin-like proteins that undergo a conformational transition for GTP-controlled oligomerization and disrupt membranes of intracellular parasites to exert their function as part of the innate immune system of mammalian cells. We apply neutron spin echo, X-ray scattering, fluorescence, and EPR spectroscopy as techniques for integrative dynamic structural biology to study the structural basis and mechanism of conformational transitions in the human GBP1 (hGBP1). We mapped hGBP1's essential dynamics from nanoseconds to milliseconds by motional spectra of sub-domains. We find a GTP-independent flexibility of the C-terminal effector domain in the µs-regime and resolve structures of two distinct conformers essential for an opening of hGBP1 like a pocket knife and for oligomerization. Our results on hGBP1's conformational heterogeneity and dynamics (intrinsic flexibility) deepen our molecular understanding relevant for its reversible oligomerization, GTP-triggered association of the GTPase-domains and assembly-dependent GTP-hydrolysis.

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.