Human guanylate-binding protein 1 (hGBP1) belongs to the superfamily of large, dynamin-related GTPases. The expression of hGBP1 is induced by stimulation with interferons (mainly interferon-γ), and it plays a role in different cellular responses to inflammatory cytokines, e.g. pathogen defence, control of proliferation, and angiogenesis. Although other members of the dynamin superfamily show a diversity of cellular functions, they share a common GTPase mechanism that relies on nucleotide-controlled oligomerization and self-activation of the GTPase. Previous structural studies on hGBP1 have suggested a mechanism of GTPase and GDPase activity that, as a critical step, involves dimerization of the large GTP-binding domains. In this study, we show that the guanine cap of hGBP1 is the key structural element responsible for dimerization, and is thereby essential for self-activation of the GTPase activity. Studies of concentration-dependent GTP hydrolysis showed that mutations of residues in the guanine cap, in particular Arg240 and Arg244, resulted in higher dissociation constants of the dimer, whereas the maximum hydrolytic activity was largely unaffected. Additionally, we identified an intramolecular polar contact (Lys62–Asp255) whose mutation leads to a loss of self-activation capability and controlled oligomer formation. We suggest that this contact structurally couples the guanine cap to the switch regions of the GTPase, translating the structural changes that occur upon nucleotide binding to a change in oligomerization and self-activation.
Structured digital abstract