title: Role of N-terminal myristylation in the structure and regulation of cAMP-dependent protein kinase.
abstract: The catalytic (C) subunit of cAMP-dependent protein kinase [protein kinase A (PKA)] is a major target of cAMP signaling, and its regulation is of fundamental importance to biological processes. One mode of regulation is N-myristylation, which has eluded structural and functional characterization so far because most crystal structures are of the non-myristylated enzyme, are phosphorylated on Ser10, and generally lack electron density for the first 13 residues. We crystallized myristylated wild-type (WT) PKA and a K7C mutant as binary (bound to a substrate peptide) and ternary [bound to a substrate peptide and adenosine-5'-(β,γ-imido)triphosphate] complexes. There was clear electron density for the entire N-terminus in the binary complexes, both refined to 2.0 Å, and K7C ternary complex, refined to 1.35 Å. The N-termini in these three structures display a novel conformation with a previously unseen helix from residues 1 to 7. The K7C mutant appears to have a more stable N-terminus, and this correlated with a significant decrease in the B-factors for the N-terminus in the myr-K7C complexes compared to the WT binary complex. The N-terminus of the myristylated WT ternary complex, refined to 2.0 Å, was disordered as in previous structures. In addition to a more ordered N-terminus, the myristylated K7C mutant exhibited a 53% increase in k(cat). The effect of nucleotide binding on the structure of the N-terminus in the WT protein and the kinetic changes in the K7C protein suggest that myristylation or occupancy of the myristyl binding pocket may serve as a site for allosteric regulation in the C-subunit.
authors.affiliation: Department of Pharmacology, University of California San Diego, San Diego, CA 92093, USA.
authors.forename: Adam C
authors.initials: AC
authors.lastname: Bastidas
authors.forename: Michael S
authors.initials: MS
authors.lastname: Deal
authors.forename: Jon M
authors.initials: JM
authors.lastname: Steichen
authors.forename: Malik M
authors.initials: MM
authors.lastname: Keshwani
authors.forename: Yurong
authors.initials: Y
authors.lastname: Guo
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D019814:Myristic Acid / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D010455:Peptides; D021122:Protein Subunits; D019814:Myristic Acid; D017868:Cyclic AMP-Dependent Protein Kinases