The αC-β4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A

Olivieri, C.; Wang, Y.; Walker, C.; Subrahmanian, M. V.; Ha, K. N.; Bernlohr, D. A.; Gao, J.; Camilloni, C.; Vendruscolo, M.; Taylor, S. S.; Veglia, G.

doi:10.1101/2023.09.12.557419

bioRxiv · 10.1101/2023.09.12.557419

The αC-β4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A

Olivieri, C.·Wang, Y.·Walker, C.·Subrahmanian, M. V.·Ha, K. N.·Bernlohr, D. A.·Gao, J.·Camilloni, C.·Vendruscolo, M.·Taylor, S. S.·Veglia, G.

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Abstract

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA). Not only this long-range synergistic action is involved in substrate recognition and fidelity, but it is also 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 integrated NMR-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of the catalytic subunit of protein kinase A (PKA-C). We found that the apoenzyme 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 C 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 C-{beta}4 loop. To experimentally validate the second excited state, we mutated F100 into alanine (F100A) and used NMR spectroscopy to characterize the structural response of the kinase to ATP and substrate binding. While the catalytic efficiency of PKA-CF100A with a canonical peptide substrate remains unaltered, this mutation rearranges the C-{beta}4 loop conformation, interrupting the structural coupling of the two lobes and abolishing the allosteric binding cooperativity of the enzyme. The highly conserved C-{beta}4 loop emerges as a pivotal element able to control the synergistic binding between nucleotide and substrate. These results may explain how mutations or insertions near or within this motif affect the function and drug sensitivity in other homologous kinases.

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Olivieri, C., Wang, Y., Walker, C., Subrahmanian, M. V., Ha, K. N., Bernlohr, D. A., Gao, J., Camilloni, C., Vendruscolo, M., Taylor, S. S., Veglia, G.. 2023-09-15. The αC-β4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A. https://doi.org/10.1101/2023.09.12.557419

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