The GTP ribose 3'OH is essential for the activation of GTP hydrolysis in trans. (a) Oligomerisation of 80 μM WT Irga6 protein was monitored by light scattering in the presence of 10 mM GTP, 2'dGTP, 3'dGTP or 2'3'ddGTP at 37°C. (b) Hydrolysis of 10 mM GTP, 2'dGTP, 3'dGTP or 2'3'ddGTP was measured in the presence of 80 μM WT Irga6 protein at 37°C. Samples were assayed by HPLC. (c) Hydrolysis of 1 mM 3'dGTP (with traces γ32P-3'dGTP) was measured in the presence of 50 μM WT Irga6 protein at 37°C. The experiment was performed with and without the addition of 1 mM unlabeled GTP, 2'dGTP, 3'dGTP or 2'3'ddGTP. Samples were assayed by TLC and autoradiography. (d) Model of the interaction between labeled 3'dGTP and unlabeled GTP in the core of the Irga6 dimer. The radioactively labeled γ-phosphate of the 3'dGTP is marked with a gray circle. The putative activatory trans interaction between the 3'OH of GTP and the γ-phosphate of 3'dGTP is represented as a dotted line.
Pawlowski et al. BMC Biology 2011 9:7 doi:10.1186/1741-7007-9-7