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

Author:

Olivieri Cristina1ORCID,Wang Yingjie12ORCID,Walker Caitlin1,Subrahmanian Manu Veliparambil1ORCID,Ha Kim N3,Bernlohr David1,Gao Jiali2ORCID,Camilloni Carlo4ORCID,Vendruscolo Michele4ORCID,Taylor Susan S56ORCID,Veglia Gianluigi12ORCID

Affiliation:

1. Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota

2. Department of Chemistry and Supercomputing Institute, University of Minnesota

3. Department of Chemistry and Biochemistry, St. Catherine University

4. Department of Chemistry, University of Cambridge

5. Department of Pharmacology, University of California at San Diego

6. Department of Chemistry and Biochemistry, University of California at San Diego

Abstract

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA’s association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of 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 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 αC-β4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-CF100A preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-β4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-β4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.

Funder

National Institute of General Medical Sciences

National Heart, Lung, and Blood Institute

Guangdong Pearl River Talent Program

National Natural Science Foundation of China-China Academy of General Technology Joint Fund for Basic Research

National Institutes of Health

Publisher

eLife Sciences Publications, Ltd

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