Tetramerization of human guanylate-binding protein 1 is mediated by coiled-coil formation of the C-terminal α-helices.

The human guanylate-binding protein 1 (hGBP1) is a large GTP-binding protein belonging to the dynamin family, a common feature of which is nucleotide-dependent assembly to homotypic oligomers. Assembly leads to stimulation of GTPase activity, which, in the case of dynamin, is responsible for scission of vesicles from membranes. By yeast two-hybrid and biochemical experiments we addressed intermolecular interactions between all subdomains of hGBP1 and identified the C-terminal subdomain, a12/13, as a new interaction site for self-assembly. a12/13 represents a stable subdomain of hGBP1, as shown by CD spectroscopy. In addition to contacts between GTPase domains leading to dimer formation, the interaction between two a12/13 subdomains, in the course of GTP hydrolysis, results in tetramer formation of the protein. With the help of CD spectroscopy we showed coiled-coil formation of two a12/13 subdomains and concentration-dependent measurements allow estimating a value for the dissociation constant of 7.3 mu m. We suggest GTP hydrolysis-driven release of the a12/13 subdomain, making it available for coiled-coil formation. Furthermore, we can demonstrate the biological relevance of hGBP1 tetramer formation in living cells by chemical cross-link experiments. Structured digital abstract and by () and by (View Interaction: , ) with by (View Interaction: , , )