In Vitro Selection of State-Specific Peptide Modulators of G Protein Signaling Using mRNA Display

Abstract

The G protein regulatory (GPR) motif is a ∼20-residue conserved domain that acts as a guanine dissociation inhibitor (GDI) for G_(i/oα) subunits. Here, we describe the isolation of peptides derived from a GPR consensus sequence using mRNA display selection libraries. Biotinylated G_(iα1), modified at either the N or C terminus, serves as a high-affinity binding target for mRNA-displayed GPR peptides. In vitro selection using mRNA display libraries based on the C terminus of the GPR motif revealed novel peptide sequences with conserved residues. Surprisingly, selected peptides contain mutations to a highly conserved Arg in the GPR motif, previously shown to be crucial for binding and inhibition activities. The dominant peptide from the selection, R6A, and a minimal 9-mer peptide, R6A-1, do not contain Arg residues yet retain high affinity (K_D = 60 and 200 nM, respectively) and specificity for the GDP-bound state of G_(iα1), as measured by surface plasmon resonance. The selected peptides also maintain GDI activity for G_(iα1), inhibiting both the exchange of GDP in GTPγS binding assays and the AlF_4^--stimulated enhancement of intrinsic tryptophan fluorescence. The kinetics of GDI activity, however, are different for the selected peptides and demonstrate biphasic kinetics, suggesting a complex mechanism for inhibition. Like the GPR motif, the R6A and R6A-1 peptides compete with G_(βγ) subunits for binding to G_(iα1), suggesting their use as activators of G_(βγ) signaling.