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 bou...

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Bibliographic Details
Published in:The Journal of biological chemistry 2003-12, Vol.278 (51), p.51825-51832
Main Authors: Adhikari, Anirban, Sprang, Stephen R.
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Animals
Binding Sites
Calorimetry
Carrier Proteins - chemistry
Carrier Proteins - metabolism
Cloning, Molecular
GTP-Binding Protein alpha Subunits, Gi-Go - chemistry
GTP-Binding Protein alpha Subunits, Gi-Go - metabolism
Guanosine 5'-O-(3-Thiotriphosphate) - metabolism
Peptide Fragments - pharmacology
Protein Binding - drug effects
Rats
Sequence Deletion
Thermodynamics
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Summary: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 Gαi1 and inhibited the binding of GTP by stabilizing the GDP-bound conformation of Gαi1. The stoichiometry, free energy, enthalpy, and dissociation constant for binding of AGS3-C to Gαi1 were determined using isothermal titration calorimetry. AGS3-C possesses two apparent high affinity (Kd ∼ 20 nm) and two apparent low affinity (Kd ∼ 300 nm) binding sites for Gαi1. Upon deletion of the C-terminal GPR motif from AGS3-C, the remaining sites were approximately equivalent with respect to their affinity (Kd ∼ 400 nm) for Gαi1. 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 Gαi1 with Kd values in the range of 1-8 μm. Although GPR1, GPR2, and GPR4 inhibited the binding of the fluorescent GTP analog BODIPY-FL-guanosine 5′-3-O-(thio)triphosphate to Gαi1, GPR3 did not. However, addition of N- and C-terminal flanking residues to the GPR3 GoLoco core increased its affinity for Gαi1 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 Gαi1 even though the amino acid sequences of these residues are not conserved among the GPR repeats.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M306300200