Dataset Information

Protein Kinase Structure and Dynamics: Role of the αC-β4 Loop.

ABSTRACT: Although the αC-β4 loop is a stable feature of all protein kinases, the importance of this motif as a conserved element of secondary structure, as well as its links to the hydrophobic architecture of the kinase core, has been underappreciated. We first review the motif and then describe how it is linked to the hydrophobic spine architecture of the kinase core, which we first discovered using a computational tool, Local Spatial Pattern (LSP) alignment. Based on NMR predictions that a mutation in this motif abolishes the synergistic high-affinity binding of ATP and a pseudo substrate inhibitor, we used LSP to interrogate the F100A mutant. This comparison highlights the importance of the αC-β4 loop and key residues at the interface between the N- and C-lobes. In addition, we delved more deeply into the structure of the apo C-subunit, which lacks ATP. While apo C-subunit showed no significant changes in backbone dynamics of the αC-β4 loop, we found significant differences in the side chain dynamics of K105. The LSP analysis suggests disruption of communication between the N- and C-lobes in the F100A mutant, which would be consistent with the structural changes predicted by the NMR spectroscopy.

SUBMITTER: Wu J

PROVIDER: S-EPMC10491255 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Protein Kinase Structure and Dynamics: Role of the αC-β4 Loop.

Wu Jian J Jonniya Nisha A NA Hirakis Sophia P SP Olivieri Cristina C Veglia Gianluigi G Kornev Alexandr P AP Taylor Susan S SS

bioRxiv : the preprint server for biology 20231016

Although the αC-β4 loop is a stable feature of all protein kinases, the importance of this motif as a conserved element of secondary structure, as well as its links to the hydrophobic architecture of the kinase core, has been underappreciated. We first review the motif and then describe how it is linked to the hydrophobic spine architecture of the kinase core, which we first discovered using a computational tool, Local Spatial Pattern (LSP) alignment. Based on NMR predictions that a mutation in ...[more]

PMID: 37693538

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Project description:Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic (C) subunit of protein kinase A (PKA). Not only this long-range synergistic action is involved in substrate recognition and fidelity, but it is likely to regulate PKA association with regulatory subunits and other binding partners. To date, a complete understanding of the molecular determinants for this intramolecular mechanism is still lacking. Here, we used an integrated NMR-restrained molecular dynamics simulations and a Markov Model to characterize the free energy landscape and conformational transitions of the catalytic subunit of protein kinase A (PKA-C). We found that the apo-enzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the tip of the αC-β4 loop. To experimentally validate the second excited state, we mutated F100 into alanine and used NMR spectroscopy to characterize the binding thermodynamics and structural response of ATP and a prototypical peptide substrate. While the activity of PKA-CF100A toward a prototypical peptide substrate is unaltered and the enzyme retains its affinity for ATP and substrate, this mutation rearranges the αC-β4 loop conformation interrupting the allosteric coupling between nucleotide and substrate. The highly conserved αC-β4 loop emerges as a pivotal element able to modulate the synergistic binding between nucleotide and substrate and may affect PKA signalosome. These results may explain how insertion mutations within this motif affect drug sensitivity in other homologous kinases.

Project description:The enthalpic and entropic components of ligand-protein binding free energy reflect the interactions and dynamics between ligand and protein. Despite decades of study, our understanding and hence our ability to predict these individual components remains poor. In recent years, there has been substantial effort and success in the prediction of relative and absolute binding free energies, but the prediction of the enthalpic (and entropic) contributions in biomolecular systems remains challenging. Indeed, it is not even clear what kind of performance in terms of accuracy could currently be obtained for such systems. It is, however, relatively straight-forward to compute the enthalpy of binding. We thus evaluated the performance of absolute enthalpy of binding calculations using molecular dynamics simulation for ten inhibitors against a member of the bromodomain family, BRD4-1, against isothermal titration calorimetry data. Initial calculations, with the AMBER force-field showed good agreement with experiment (R2 = 0.60) and surprisingly good accuracy with an average of root-mean-square error (RMSE) = 2.49 kcal mol-1. Of the ten predictions, three were obvious outliers that were all over-predicted compared to experiment. Analysis of various simulation factors, including parameterization, buffer concentration and conformational dynamics, revealed that the behaviour of a loop (the ZA loop on the periphery of the binding site) strongly dictates the enthalpic prediction. Consistent with previous observations, the loop exists in two distinct conformational states and by considering one or the other or both states, the prediction for the three outliers can be improved dramatically to the point where the R2 = 0.95 and the accuracy in terms of RMSE improves to 0.90 kcal mol-1. However, performance across force-fields is not consistent: if OPLS and CHARMM are used, different outliers are observed and the correlation with the ZA loop behaviour is not recapitulated, likely reflecting parameterization as a confounding problem. The results provide a benchmark standard for future study and comparison.

Dataset Information

Protein Kinase Structure and Dynamics: Role of the αC-β4 Loop.

Publications

Protein Kinase Structure and Dynamics: Role of the αC-β4 Loop.

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