title: A peptide core motif for binding to heterotrimeric G protein alpha subunits.
abstract: Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to Galpha(i1). The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of Galpha(i1) and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with Galpha subunits representing all four G protein classes, acting as a core motif for Galpha interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (Galpha(i/0)-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to Galpha(i1-3) in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the Galpha subunits and excludes association with Gbetagamma. R6A-Galpha(i1) complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg(2+), suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using Galpha(i1)/Galpha(s) chimeras identify two regions of Galpha(i1) (residues 1-35 and 57-88) as determinants for strong R6A-G(ialpha1) interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, 91125, USA.
authors.forename: William W
authors.initials: WW
authors.lastname: Ja
authors.forename: Anirban
authors.initials: A
authors.lastname: Adhikari
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Austin
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Sprang
authors.forename: Richard W
authors.initials: RW
authors.lastname: Roberts
mesh_terms: D020816:Amino Acid Motifs; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry*; D005822:Genetic Vectors; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D047468:Immunoprecipitation; D046911:Macromolecular Substances / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008957:Models, Genetic; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D044385:GTP-Binding Protein alpha Subunits; D046911:Macromolecular Substances; D010455:Peptides; C508077:R6A-1 peptide; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D014357:Trypsin; D008274:Magnesium