Globally correlated conformational entropy underlies positive and negative cooperativity in a kinase's enzymatic cycle

Abstract

Enzymes accelerate the rate of chemical transformations by reducing the activation barriers of uncatalyzed reactions. For signaling enzymes, substrate recognition, binding, and product release are often rate-determining steps in which enthalpy-entropy compensation plays a crucial role. While the nature of enthalpic interactions can be inferred from structural data, the molecular origin and role of entropy in enzyme catalysis remains poorly understood. Using thermocalorimetry, NMR, and MD simulations, we studied the conformational landscape of the catalytic subunit of cAMP-dependent protein kinase A, a ubiquitous phosphoryl transferase involved in a myriad of cellular processes. Along the enzymatic cycle, the kinase exhibits positive and negative cooperativity for substrate and nucleotide binding and product release. We found that globally coordinated changes of conformational entropy activated by ligand binding, together with synchronous and asynchronous breathing motions of the enzyme, underlie allosteric cooperativity along the kinase's cycle.

Fig. 1

Conformational transition of PKA-C during turnover. a Superposition of the X-ray crystal structures of PKA-C in the apo (PDB code: 4NTS), binary complex (ATPγN-bound, PDB code: 1BKX), ternary complex (ATPγN and PKS_5-24, PDB code: 4DG0), ternary/exit complex (ADP and pPKS bound, PDB code: 4IAF), and binary (ADP-bound, PDB code: 4NTT). Dotted arrows indicate the major domains involved in large amplitude motions determining opening and closing of the nucleotide site and substrate hub. b Principal component analysis (PCA) with the two main components indicating the structural transitions in the crystal structures of PKA-C for different ligated states, where PC1 and PC2 involve distinct collective motions throughout the protein (illustrated in Supplementary Fig. 1). c CONCISE plot showing the probability distribution curves of the methyl chemical shifts for the different states along the open-to-close trajectories. d Changes in the free energy of binding (ΔΔG) determined by ITC (blue) measurements (mean ± SD, n = 3) compared with the ΔΔG obtained from the CONCISE analysis (red) that estimates the relative population of the bound states. The CONCISE data are obtained from probability density distributions of the NMR chemical shifts for the ILV methyl groups of PKA-C, where the error bar reports the 90% confidence interval. The data of the CONCISE plots are reported in the Source Data file

Fig. 2

Changes of conformational entropy of PKA-C along the coordinate of reactions. Ribbon representation of the different ligated conformations of PKA-C along the coordinate of reactions as obtained from X-ray crystallography: apo (PDB code: 4NTS), binary complex (ATPγN-bound, PDB code: 1BKX), ternary complex (ATPγN and PKS_5–24 bound, PDB code: 4DG0), ternary/exit complex (ADP and pPKS_5-24 bound, PDB code: 4IAF), and binary (ADP-bound, PDB code: 4NTT). The methyl group O² obtained for the different forms are indicated with a colored gradient from the most rigid (blue) to the most mobile (red). The ligands are omitted from the figure for clarity. For each transition, we indicated the signs for total free energy (green), enthalpy (red), and entropic penalty (blue) as obtained from ITC measurements as well as the average sign for conformational entropy (purple) reflected by ΔO² from NMR measurements

Fig. 3

Correlated motions orchestrate positive and negative cooperativity. a Covariance plot indicating the coordinated changes of methyl group order parameters (O²) throughout the entire kinase. The diagonal peaks in the plots represent the assigned methyl groups, while cross peaks indicate the degree of correlation of the methyl group O² using Pearson coefficient as a metric. Methyl group dynamics changes are considered coordinated when the Pearson coefficient is greater than 0.9. b Network plot connecting the methyl groups the highest values of covariance. c Mapping of the methyl groups that show a significant difference in order parameters upon forming the ternary complexes, i.e., PKA-C/ATPγN/PKS_5–24 and PKA-C/ADP/pPKS_5–24, from the binary complexes bound with nucleotide, respectively (see Supplementary Fig. 12)

Fig. 4

Conformational entropy analysis of PKA-C based on MD simulations. Conformational entropy analysis of the main chain and side chains of the Apo, ATP, ATP + substrate and the ADP + phospho-product forms of PKA-C. The altered entropy of the enzyme upon binding ATP primes the enzyme for substrate binding, whereas the change of entropy in the ADP + phospho-product state primes the enzyme for product release. Changes in entropy are distributed globally through the enzyme

Fig. 5

Synchronous and asynchronous motions of PKA-C along reaction. a Conformational exchange rates obtained using methyl CPMG experiments mapped onto the X-ray crystal structures: apo (PDB code: 4NTS), binary complex (ATPγN-bound, PDB code: 1BKX), ternary complex (ATP and PKS_5–24, PDB code: 4DG0), ternary/exit complex (ADP and pPKS bound, PDB code: 4IAF), and binary (ADP-bound, PDB code: 4NTT). b DyCorr maps of the different states of the kinase, showing the synchronization of motion upon nucleotide binding and the asynchronous dynamics in the ternary exit complex and ADP bound