Additional file 11.
Asp164 and Arg159 participate in oligomerisation. For the construction of the Irga6 dimer model a rigid crystal structure was used. In the model the side chains of the Arg159 residues of the two subunits collide. Arg159 is located close to Asp164 on the other subunit. Asp164 forms the bottom of a pocket, derived from two loops. One loop is located between Glu77 and Ser80 and contains a part of the G1-motif. The other loop is located between Ile155 and Asn163. The conformation of Arg159 is relatively unconstrained . A conformational change may occur during complex formation, reorienting Arg159 and inserting the side chain into the pocket on the opposed molecule to form a salt bridge with Asp164 in trans. Arg159 is part of the catalytic interface (Figure 1a). Consistent with this, mutations of Arg159 had deleterious effects on oligomerisation (Additional file 12). Asp164 is not solvent exposed, but withdrawn from the surface of the protein at the bottom of a pocket. It is therefore striking that even a mild mutation like D164N prevented oligomerisation (Additional file 13). (a and b) View of the nucleotide-binding region. (a) The Irga6 dimer model (Figure 4) is shown. Arg159, Asp164 (cyan subunit) and Arg159 (magenta subunit) are shown. (b) A molecule of Irga6-M173A  is shown. Asp164 and the molecular surface formed by the residues Glu77, Thr78, Gly79, Ser80, Ile155, Ser156, Ala157, Thr158, Arg159, Phe160, Lys161, Lys162 and Asn163 are shown. (c) Oligomerisation of 80 μM WT or mutant Irga6 proteins was monitored by light scattering in the presence of 10 mM GTP at 37°C. (d) Hydrolysis of 10 mM GTP (with traces α32P-GTP) was measured in the presence of 80 μM WT or mutant Irga6 proteins at 37°C. Samples were assayed by TLC and autoradiography.
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Pawlowski et al. BMC Biology 2011 9:7 doi:10.1186/1741-7007-9-7