A Residue outside the Binding Site Determines the Gα Binding Specificity of GoLoco Motifs

GoLoco motif-containing proteins regulate the nucleotide-binding state of Gα proteins in various signaling pathways. As guanine nucleotide dissociation inhibitors (GDIs), they bind Gα·GDP and inhibit GDP to GTP exchange. GoLoco proteins show binding selectivity toward different members of the Gα family. Although the Gα_i1·GDP/RGS14 crystal structure explains the specific binding selectivity of the RGS14 GoLoco domain well, the mechanism of selective binding has not been understood for the more general features of short GoLoco domains found in tandem arrays in proteins like GPSM2/LGN/ dPins and GPSM1/AGS3. We explored the mechanism of differential interactions of GoLoco protein LGN with hGα_i3 and hGα_o. By combining mutagenesis experiments and molecular dynamics simulations, we identified a residue (Asp229 in hGα_i3) away from the binding interface that remarkably affects the interaction between LGN and hGα_i/o. A negatively charged residue at this position is required for high binding affinity. This affinity regulation mechanism was further verified by the cases of hGα_i2 and dGα_o, suggesting that this pathway is conserved among members of the Gα family.