{"doi":"10.1101/2023.05.29.542785","title":"Understanding ATP binding to DosS catalytic domain with a short ATP-lid","abstract":"ABSTRACT DosS is a heme-sensor histidine kinase that responds to redox-active stimuli in mycobacterial environments by triggering dormancy transformation. Sequence comparison of the catalytic ATP-binding (CA) domain of DosS to other well-studied histidine kinases suggests that it possesses a rather short ATP-lid. This feature has been thought to inhibit DosS kinase activity by blocking ATP binding in the absence of interdomain interactions with the dimerization and histidine phospho-transfer (DHp) domain of full-length DosS. Here, we use a combination of computational modeling, structural biology, and biophysical studies to re-examine ATP-binding modalities in DosS’s CA domain. We show that the closed lid conformation observed in protein crystal structures of DosS CA is caused by the presence of a zinc cation in the ATP binding pocket that coordinates with a glutamate residue on the ATP-lid. Furthermore, circular dichroism (CD) studies and comparisons of DosS CA crystal structure with its AlphaFold model and homologous DesK reveal that a key N-box alpha-helix turn of the ATP pocket manifests as a random coil in the zinc-coordinated protein crystal structure. We note that this closed lid conformation and the random-coil transformation of an N-box alpha-helix turn are artifacts arising from the millimolar zinc concentration used in DosS CA crystallization conditions. In contrast, in the absence of zinc, we find that the short ATP-lid of DosS CA has significant conformational flexibility and can bind ATP ( K d = 53 ± 13 μM). We conclude that DosS CA is almost always bound to ATP under physiological conditions (1-5 mM ATP, sub-nanomolar free zinc) in the bacterial environment. Our findings elucidate the conformational adaptability of the short ATP-lid, its relevance to ATP binding in DosS CA and provide insights that extends to 2988 homologous bacterial proteins containing such ATP-lids.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":407919,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9542,"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":1186913,"name":"Peter Windsor","orcid":"0000-0001-5629-3517","position":1,"is_corresponding":false},{"id":1187214,"name":"Elizabeth M. Smithwick","orcid":null,"position":2,"is_corresponding":false},{"id":103757,"name":"Ke Shi","orcid":"0000-0003-4175-3714","position":3,"is_corresponding":false},{"id":103759,"name":"Hideki Aihara","orcid":"0000-0001-7508-6230","position":4,"is_corresponding":false},{"id":1076709,"name":"Anoop R. Damodaran","orcid":"0000-0002-2094-9956","position":5,"is_corresponding":false},{"id":1076710,"name":"Ambika Bhagi‐Damodaran","orcid":"0000-0002-4901-074X","position":6,"is_corresponding":false},{"id":376212,"name":"Grant Larson","orcid":"0000-0003-3308-1591","position":0,"is_corresponding":true}],"reference_count":46,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T01:21:14.562812Z","pmid":"37398500","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":[]}