{"doi":"10.7554/elife.91506","title":"The αC-β4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A","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-C F100A 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.","journal":"eLife","year":2023,"id":364706,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":7,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9484,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":231459,"name":"Yingjie Wang","orcid":"0000-0001-9800-8163","position":1,"is_corresponding":false},{"id":585853,"name":"Caitlin Walker","orcid":"0000-0003-0977-8276","position":2,"is_corresponding":false},{"id":585854,"name":"Manu Veliparambil Subrahmanian","orcid":"0000-0002-1374-2797","position":3,"is_corresponding":false},{"id":1120291,"name":"Kim N. Ha","orcid":null,"position":4,"is_corresponding":false},{"id":484734,"name":"David Bernlohr","orcid":"0000-0001-6969-9129","position":5,"is_corresponding":false},{"id":231460,"name":"Jiali Gao","orcid":"0000-0003-0106-7154","position":6,"is_corresponding":false},{"id":860733,"name":"Carlo Camilloni","orcid":"0000-0002-9923-8590","position":7,"is_corresponding":false},{"id":268831,"name":"Michele Vendruscolo","orcid":"0000-0002-3616-1610","position":8,"is_corresponding":false},{"id":232971,"name":"Susan S. Taylor","orcid":"0000-0002-7702-6108","position":9,"is_corresponding":false},{"id":364784,"name":"Gianluigi Veglia","orcid":"0000-0002-2795-6964","position":10,"is_corresponding":false},{"id":364783,"name":"Cristina Olivieri","orcid":"0000-0001-6957-6743","position":0,"is_corresponding":true}],"reference_count":88,"raw_metadata":null,"created_at":"2026-07-19T01:14:41.218128Z","pmid":"38913408","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}