Evidence for a Second, High Affinity Gβγ Binding Site on Gαi1(GDP) Subunits

It is well known that G alpha(i1)(GDP) binds strongly to G beta gamma subunits to form the G alpha(i1)(GDP)-G beta gamma heterotrimer, and that activation to G alpha(i1)(GTP) results in conformational changes that reduces its affinity for G beta gamma subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that G alpha(i1)(GDP) can bind a second G beta gamma subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated G alpha(i1) and G beta gamma subunits. Also, we find that phospholipase C beta 2, an effector of G beta gamma, does not compete with the second binding site implying that effectors can be bound to the G alpha(i1)(GDP)-(G beta gamma)2 complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on G alpha(i1) competes with binding of the second G beta gamma subunit. Injection of this peptide into cultured cells expressing eYFP-G alpha(i1)(GDP) and eCFP-G beta gamma reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.