Figure 6

The nucleotide base is part of the catalytic interface. (a and b) View of the nucleotide-binding region. The Irga6 dimer model (Figure 4) is shown. Glu77, Ser132 (magenta), Asp186 (cyan), of WT Irga6, with two GppNHp nucleotides (a) and modeled Asn186 (cyan), of Irga6-D186N, with two XTP nucleotides (b) are shown. The interactions of Asp186 with GppNHp and of Asn186 with XTP are represented by dotted lines. (c) Oligomerisation of 80 μM Irga6-D186N protein was monitored by light scattering in the presence of 10 mM GTP at 37°C. The experiment was performed with and without the addition of 1 mM XTP. (d) Hydrolysis of 10 mM GTP (with traces α³²P-GTP) was measured in the presence of 80 μM Irga6-D186N protein at 37°C. The experiment was performed with and without the addition of 1 mM XTP. Samples were assayed by TLC and autoradiography.