Influence of N‑Myristylation and Ligand Binding on the Flexibility of the Catalytic Subunit of Protein Kinase A

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.