Structural Basis for G α i Activation by a Non‐receptor Protein

Abstract

The recent structural elucidation of the β2-AR/ Gαs complex is a landmark in the understanding of the molecular mechanisms of trimeric G protein activation. What remains unknown is whether this mechanism is conserved in another subset of G protein activators which are non-receptor proteins. Non-receptor GEFs have emerged as important signaling components in diverse cellular processes and their involvement in disease makes them attractive therapeutic targets. Here we used nuclear magnetic resonance (NMR) to study residue-level conformational changes in Gαi3 upon binding of GIV, a non-receptor GEF. Backbone signals for ~75% of Gαi3 residues were assigned with high confidence and changes in them analyzed upon GIV binding in solution. Signal perturbations were observed not only in a previously predicted binding region (SwII/α3) but also in the nucleotide binding pocket and a GPCR binding loop. Based on this we carried out an extensive site-directed mutagenesis study from which we conclude that GIV binding on Gαi does not overlap with the GPCR binding region and that it induces conformational changes in the nucleotide binding pocket allosterically. These changes in conformation are only partially overlapping with those observed upon GPCR activation. Taken together these results indicate that GIV and GPCRs utilize markedly different molecular mechanisms to activate G proteins. These new insights indicate that at least some non-receptor GEFs can be specifically targeted without affecting GPCR-G protein coupling and also suggest why receptor and non-receptor GEFs have different potency. Support: NIH R01GM108733