Dimerization and its role in GMP formation by human guanylate binding proteins

Abstract

The mechanism of oligomerization and its role in the regulation of activity in large GTPases are not clearly understood. Human guanylate binding proteins (hGBP-1 and 2) belonging to large GTPases have the unique feature of hydrolyzing GTP to a mixture of GDP and GMP with unequal ratios. Using a series of truncated and mutant proteins of hGBP-1, we identified a hydrophobic helix in the connecting region between the two domains that plays a critical role in dimerization and regulation of the GTPase activity. The fluorescence with 1-8-anilinonaphthalene sulfonate and circular dichroism measurements together suggest that in the absence of the substrate analog, the helix is masked inside the protein but becomes exposed through a substrate-induced conformational switch, and thus mediates dimerization. This is further supported by the intrinsic fluorescence experiment, where Leu(298) of this helix is replaced by a tryptophan. Remarkably, the enzyme exhibits differential GTPase activities depending on dimerization; a monomer produces only GDP, but a dimer gives both GDP and GMP with stimulation of the activity. An absolute dependence of GMP formation with dimerization demonstrates a cross talk between the monomers during the second hydrolysis. Similar to hGBP-1, hGBP-2 showed dimerization-related GTPase activity for GMP formation, indicating that this family of proteins follows a broadly similar mechanism for GTP hydrolysis.