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.

Figure 1

Sites of FM conjugation in the C-subunit. (A) The structure of the N-myristylated C-subunit of PKA (PDB: 4DFX(16)) is displayed in cartoon representation with the small lobe (1–126) in gray, large lobe (127–300) in olive, C-tail (301–350) in red, inhibitor peptide in cyan, myristic acid in orange as a stick representation, and sites of mutation in yellow as a stick and surface representation. (B) A stereoview of the A-helix and myristate pocket is shown in cartoon representation with the regions of the protein and sites of FM conjugation depicted and colored as in (A). Several other residues within or near the myristate pocket are also shown in stick representation, and myristic acid is shown in sphere representation. (C) A stereoview of regions of the protein near the other sites of labeling is shown in cartoon representation with the regions of the protein and sites of fluorescent labeling depicted and colored as in (A).

Figure 2

Myristylation stabilizes the N-terminus of the C-subunit, measured via time-resolved fluorescence anisotropy and MD simulations. (A) Anisotropy decay of FM-K7C in myristylated/apo state (blue), nonmyristylated/apo state (red), myristylated/ternary state (orange), and nonmyristylated/ternary state (green). IRF is the instrument response function. (B) Anisotropy decay of FM-K16C with the different states colored as in (A). (C) The root mean squared fluctuation (RMSF) values averaged from six replicate MD simulations of myristylated and nonmyristylated configurations with and without ligands, plotted in angstroms for the A-helix residues (2–30).

Figure 3

Myristylation and ligand binding produces altered mobility of the myristate pocket. (A) Anisotropy decay of FM-N99C in myristylated/apo state (blue), nonmyristylated/apo state (red), myristylated/ternary state (orange), and nonmyristylated/ternary state (green). IRF is the instrument response function. (B–D) RMSF values of the main-chain atoms from MD simulations for residues 90–105 (B), 150–160 (C), and 300–310 (D).

Figure 4

Effects of myristylation and ligand binding on the mobility of the C-tail. (A) Anisotropy decay of FM-S325C in myristylated/apo state (blue), nonmyristylated/apo state (red), myristylated/ternary state (orange), and nonmyristylated/ternary state (green). IRF is the instrument response function. (B) RMSF values for the backbone atoms from the MD simulations near the S325 residue. (C) Anisotropy decay of FM-C343 with the different states colored as in (A). (D) The RMSF values of the backbone atoms near the C343 residue.

Figure 5

PCA analysis of the MD simulations of the C-subunit. The first two principal components were calculated from the backbone atoms of all MD simulations. (A) Structural ensembles of the backbone atoms for the nonmyristylated C-subunit in apo and ternary states and myristylated C-subunit in apo and ternary states are shown. (B) Major movements associated with PC1 and PC2 are depicted on the C-subunit. PC1 is associated with a vertical movement of the N-terminus and Gly-rich loop, and PC2 involves a rotation of the N-terminus and Gly-rich loop. (C) Changes in the Gly-rich loop are shown for PC1 (gray) and PC2 (olive) through the projections aligned with the crystal structure 4DFX (black).

Figure 6

Possible modes of crosstalk between the myristate pocket and active site. (A) The N-lobe is gray, C-lobe and C-tail are olive, the myristic acid group is shown in sphere representation in orange, and ATP is shown in sphere representation and colored by element. Several residues that may mediate crosstalk between the myristate pocket and active site are shown in stick representation or stick and surface representation. Some residues in the myristic acid pocket are not labeled for viewing clarity but are labeled in Figure 1B. (B) The protein is colored as in (A). ATP is shown in stick representation. The regulatory and catalytic spines (R-spine and C-spine) are shown in stick and surface representation in red and yellow, respectively. (C) The C-spine and R-spine are colored and depicted as in (B), and one connection between the F-helix and R-spine is shown.