Dynamically committed, uncommitted, and quenched states encoded in protein kinase A revealed by NMR spectroscopy

Abstract

Protein kinase A (PKA) is a ubiquitous phosphoryl transferase that mediates hundreds of cell signaling events. During turnover, its catalytic subunit (PKA-C) interconverts between three major conformational states (open, intermediate, and closed) that are dynamically and allosterically activated by nucleotide binding. We show that the structural transitions between these conformational states are minimal and allosteric dynamics encode the motions from one state to the next. NMR and molecular dynamics simulations define the energy landscape of PKA-C, with the substrate allowing the enzyme to adopt a broad distribution of conformations (dynamically committed state) and the inhibitors (high magnesium and pseudosubstrate) locking it into discrete minima (dynamically quenched state), thereby reducing the motions that allow turnover. These results unveil the role of internal dynamics in both kinase function and regulation.

Fig. 1.

Thermodynamic and NMR analysis of PKA-C. (A) Thermodynamics of PKA-C binding to substrate and inhibitor, with or without ADP. The binding of PLN_1–20 is dominated by favorable entropy, whereas PKI_5–24 is enthalpy driven, overcoming an entropic penalty. (B) Melting measurements showed that PKI_5–24 confers the greatest thermostability to PKA-C as represented by the shift in T_m (relative to apo-PKA-C, ΔT_m). High Mg⁺² confers slightly higher stability to each complex. (C–E) Correlation of chemical shift perturbations (Δδ) in PKA-C between the different forms. The majority of perturbation occurs upon nucleotide binding (C), whereas formation of the ternary complexes were quite similar to one another (D and E). (F) Linearity of chemical shifts between the apo form and the ternary form (AMP-PNP/PKI_5–24 with high Mg²⁺) is observed, indicating that the enzyme opens and closes on a fast timescale. (G) Enzymes views from the active site surface formed by the small and large lobes.

Fig. 2.

Backbone dynamics of PKA-C in different ternary complexes. Mapping of (A) fast and (B) slow backbone dynamics show that, upon inhibition with PKI_5–24 and with high Mg²⁺, a decrease of picosecond to millisecond dynamics occurs throughout the backbone. For the comparison, the previously published dynamics of PKA-C with the substrate PLN_1–20 is shown (7).

Fig. 3.

Comparison of MD simulations for PKA-C. (A) Global motions suggested by PCA analysis of MD trajectories correspond to opening and closing of the active site (PC1), which compared well with the distances between residues S53 and G186 in crystal structures of open (1CMK), intermediate (1BX6), and closed (1ATP) conformations. (B) A map of the interatomic distances vs. the PC1 from MD simulations indicate that PKA-C accessed the major crystallographic conformations frequently, except in the presence of inhibitors.

Fig. 4.

The energy landscape of PKA-C is modulated by ligand binding. The apo state is dynamically uncommitted, having dynamics which are not tuned to turnover. Nucleotide binding induces motions which are synchronized to turnover (dynamically committed) and are persistent in the ternary complex with substrate. However, PKI_5–24, or excess Mg²⁺ and PKI_5–24, induces favorable enthalpy which lowers the energy of one or more conformational states and raises the energy barriers in the conformational landscape. This phenomenon hinders conformational fluctuations, inhibits turnover, and results in a dynamically quenched enzyme.