title: Structural basis for peptide binding in protein kinase A. Role of glutamic acid 203 and tyrosine 204 in the peptide-positioning loop.
abstract: For optimal activity the catalytic subunit of cAMP-dependent protein kinase requires a phosphate on Thr-197. This phosphate anchors the activation loop in the proper conformation and contributes to catalytic efficiency by enhancing the phosphoryl transfer rate and increasing the affinity for ATP (1). The crystal structure of the catalytic subunit bound to ATP, and the inhibitor peptide, IP20, highlights the contacts made by the Thr-197 phosphate as well as the role adjacent residues play in contacting the substrate peptide. Glu-203 and Tyr-204 interact with arginines in the consensus sequence of PKA substrates at the P-6 and P-2 positions, respectively. To assess the contribution that each residue makes to peptide recognition, the kinetic properties of three mutant proteins (E203A, Y204A, and Y204F) were monitored using multiple peptide substrates. The canonical peptide substrate, Kemptide, as well as a longer 9-residue peptide and corresponding peptides with alanine substitutions at the P-6 and P-2 positions were used. While the effect of Glu-203 is more localized to the P-6 site, Tyr-204 contributes to global peptide recognition. An aromatic hydrophobic residue is essential for optimal peptide recognition and is conserved throughout the protein kinase family.
authors.affiliation: Howard Hughes Medical Institute, the Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0654, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Moore
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Adams
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
mesh_terms: D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D018698:Glutamic Acid; D007700:Kinetics; D051379:Mice; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D021122:Protein Subunits; D013329:Structure-Activity Relationship; D014443:Tyrosine
chemical_list: D010455:Peptides; D021122:Protein Subunits; D018698:Glutamic Acid; D014443:Tyrosine; D017868:Cyclic AMP-Dependent Protein Kinases