title: Influence of N-myristylation and ligand binding on the flexibility of the catalytic subunit of protein kinase A.
abstract: The catalytic (C) subunit of protein kinase A is regulated in part by cotranslational N-myristylation and ligand binding. Using a combination of time-resolved fluorescence anisotropy and molecular dynamics (MD) simulations, we characterized the effect of N-myristylation and ligand binding on C-subunit dynamics. Five single-site cysteine-substitution mutants of the C-subunit were engineered with and without N-terminal myristylation and labeled with fluorescein maleimide, and time-resolved fluorescence anisotropy decays were measured to assess the flexibility of the labeled regions in the presence and absence of ligands. A parallel set of in silico experiments were performed to complement the experimental findings. These experiments showed that myristylation produces both local and global effects on C-subunit dynamics. The local effects include stabilization of the N-terminus and myristate pocket, and the global effects include small increases in mobility along the C-tail at residue C343. Additionally, ligand binding was associated with an increase in mobility of the myristate binding pocket for both the myristylated and nonmyristylated enzyme on the basis of both the experimental and MD results. Also, MD simulations suggest that the myristylated protein exhibits increased dynamics when bound to ligands compared to the nonmyristylated protein.
authors.affiliation: Department of Pharmacology, ‡Department of Chemistry and Biochemistry, ⊥San Diego Supercomputer Center, and ¶Howard Hughes Medical Institute, University of California , San Diego, California 92093, United States.
authors.forename: Adam C
authors.initials: AC
authors.lastname: Bastidas
authors.forename: Levi C
authors.initials: LC
authors.lastname: Pierce
authors.forename: Ross C
authors.initials: RC
authors.lastname: Walker
authors.forename: David A
authors.initials: DA
authors.lastname: Johnson
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
mesh_terms: D000215:Acylation; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D005454:Fluorescence Polarization; D008024:Ligands; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D019814:Myristic Acid / Q000737:chemistry*; D011487:Protein Conformation / Q000187:drug effects*
chemical_list: D008024:Ligands; D019814:Myristic Acid; D017868:Cyclic AMP-Dependent Protein Kinases