Thermodynamic characterization of the binding of activator of G protein signaling 3 (AGS3) and peptides derived from AGS3 with Gαi1

Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) that contains four G protein regulatory (GPR) or GoLoco motifs in its C-terminal domain. The entire C-terminal domain (AGS3-C) as well as certain peptides corresponding to individual GPR motifs of AGS3 bound to Galpha(i1) and inhibited the binding of GTP by stabilizing the GDP-bound conformation of Galpha(i1). The stoichiometry, free energy, enthalpy, and dissociation constant for binding of AGS3-C to Galpha(i1) were determined using isothermal titration calorimetry. AGS3-C possesses two apparent high affinity (K(d)similar to 20 nM) and two apparent low affinity (K-d similar to 300 nM) binding sites for Galpha(i1). Upon deletion of the C-terminal GPR motif from AGS3-C, the remaining sites were approximately equivalent with respect to their affinity (K-d similar to 400 nM) for Galpha(i1). Peptides corresponding to each of the four GPR motifs of AGS3 (referred to as GPR1, GPR2, GPR3, and GPR4, respectively, going from N to C terminus) bound to Galpha(i1) with K-d values in the range of 1-8 muM. Although GPR1, GPR2, and GPR4 inhibited the binding of the fluorescent GTP analog BODIPY-FL-guanosine 5'-3-O-(thio)triphosphate to Galpha(i1), GPR3 did not. However, addition of N- and C-terminal flanking residues to the GPR3 GoLoco core increased its affinity for Galpha(i1) and conferred GDI activity similar to that of AGS3-C itself. Similar increases were observed for extended GPR2 and extended GPR1 peptides. Thus, while the tertiary structure of AGS3 may affect the affinity and activity of the GPR motifs contained within its sequence, residues outside of the GPR motifs strongly potentiate their binding and GDI activity toward Galpha(i1) even though the amino acid sequences of these residues are not conserved among the GPR repeats.