Allostery and binding cooperativity of the catalytic subunit of protein kinase A by NMR spectroscopy and molecular dynamics simulations

Abstract

The catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is an exquisite example of a single molecule allosteric enzyme, where classical and modern views of allosteric signaling merge. In this chapter, we describe the mapping of PKA-C conformational dynamics and allosteric signaling in the free and bound states using a combination of NMR spectroscopy and molecular dynamics simulations. We show that ligand binding affects the enzyme's conformational dynamics, shaping the free-energy landscape toward the next stage of the catalytic cycle. While nucleotide and substrate binding enhance the enzyme's conformational entropy and define dynamically committed states, inhibitor binding attenuates the internal dynamics in favor of enthalpic interactions and delineates dynamically quenched states. These studies support a central role of conformational dynamics in many aspects of enzymatic turnover and suggest future avenues for controlling enzymatic function.

Fig. 1

Architecture of PKA-C. (A) Secondary structure, domains, conserved motifs (see asterisks), and ligand-binding regions of PKA-C. (B) Conformational changes observed in PKA-C by X-ray crystallography. The major conformational states are defined by the angle between the large and small lobes and by the S53/G186Cα distance (open, intermediate, and closed conformational states). The average angles and distances are calculated from open (PDB: 3O7L, 1CMK, 1CTP, 1J3H, and 2QVS), intermediate (PDB: 1BKX, 1BX6, 1STC, 1JLU, 1RE8, 1REK, 3DND, 3DNE, 3IDB, and 3IDC), and closed (PDB: 1JBP, 1ATP, 1APM, 1YDS, 1YDR, and 1YDT) X-ray structures.

Fig. 2

Chemical perturbations upon ligand binding for PKA-C^WT and PKA-C^Y204A. The binding of nucleotide and substrate indicates attenuated local and long-range (allosteric) perturbations for PKA-C^Y204A.

Fig. 3

Thermocalorimetric parameters for the binding of the substrate PLN_1–20 and inhibitor PKI_5–24 to PKA-C. (A) Binding energy for substrate and inhibitor to the apo or nucleotide-bound PKA-C. PLN_1–20 is entropy driven, while PKI_5–24 binding is enthalpy driven. (B) Thermal melting of PKA-C complexes. A small increase in thermal stability (relative to the apo enzyme melting temperature, ΔT) is present upon nucleotide or substrate binding under low and high Mg²⁺ concentration. A substantial increase in stability is measured in the presence of PKI_5–24 and high Mg²⁺.

Fig. 4

Linearity of chemical changes among different forms of PKA-C. (A) Linearly correlated chemical shift changes in different regions of the small and large lobes. The two extremes of the correlations are the apo and the super-inhibited ternary complex of PKA-C, suggesting a fast conformational exchange between closed and open conformational states. (B) The location of these representative residues included regions near the active site and allosteric sites as far as 20 Å (W302) away.

Fig. 5

Free-energy landscape of PKA-C and ligand binding. Ligand binding defines the conformational equilibrium of the kinase, skewing the populations in a dynamically uncommitted (apo form) basin with motions not synchronous to turnover, dynamically committed basin (nucleotide-bound and ternary complex with substrate) with motions synchronous with turnover, and dynamically quenched basin (inhibited complexes) with attenuated motions and low free energy.

Fig. 6

Schematic for the formation of the catalytically competent ternary complex. The apo enzyme constitutes a dynamically uncommitted state, with its C-spine (red) disengaged between the two lobes. The nucleotide acts as an allosteric effector which is sensed throughout the enzyme. This binding event completes the C-spine architecture and induces a dynamically committed state. Fluctuations between open and closed conformations take place at a time scale which is synchronous with turnover. These fluctuations persist in the ternary complex with substrate, limiting the rate of catalytic turnover.

Fig. 7

MD simulations of PKA-C ternary complexes with PLN_1–20 or PKI_5–24. All plots indicate ternary complexes with PLN_1–20 (red) or PKI_5–24 (black). (A) Backbone sub-nanosecond RMSF of PKA-C ternary complexes. Strong (red) and weak (green) hydrogen bonding networks which persisted during the simulations when PKI_5–24 (B) or PLN_1–20 (C) was present. (D) RMSF of the peptide backbone of PLN_1–20 or PKI_5–24. (E) The structure and atom number of ATP, and (F) RMSF of ATP during simulations with the substrate or inhibitor.

Fig. 8

MD simulations of PKA-C. (A) PCA analysis of the MD trajectories indicated two orthogonal motions between the small and large lobes which described ~50% of the variance: an opening/closing motion (PC1) and a shearing between the lobes (PC2). (B) The occlusion of the active site was probed using the distance between S53 and G186 C^α atoms and monitored with PC1 to assess active site accessibility. (C) 2D plots indicated that inhibitors restrict the conformational space accessible by PKA-C, while the other forms accessed these states more frequently.

Fig. 9

Proposed mutual conformational selection mechanism between PKA-C and PLN. Both PKA-C and PLN undergo preexisting equilibria between major conformational states. PKA-C fluctuates between open and closed conformations, induced by nucleotide binding. PLN undergoes conformational fluctuations between various degrees of folded and unfolded conformations. These equilibria influence one another upon interaction, leading to a mutual adaptation and allowing the proteins to reach a minimum in the free-energy landscape of the complex.