{"doi":"10.1021/acs.biochem.3c00391","title":"Full-Length NAD<sup>+</sup>-I Riboswitches Bind a Single Cofactor but Cannot Discriminate against Adenosine Triphosphate","abstract":"Bacterial riboswitches are structured RNAs that bind small metabolites to control downstream gene expression. Two riboswitch classes have been reported to sense nicotinamide adenine dinucleotide (NAD+), which plays a key redox role in cellular metabolism. The NAD+-I (class I) riboswitch stands out because it comprises two homologous, tandemly arranged domains. However, previous studies examined the isolated domains rather than the full-length riboswitch. Crystallography and ligand binding analyses led to the hypothesis that each domain senses NAD+ but with disparate equilibrium binding constants (KD) of 127 μM (domain I) and 3.4 mM (domain II). Here, we analyzed individual domains and the full-length riboswitch by isothermal titration calorimetry to quantify the cofactor affinity and specificity. Domain I senses NAD+ with a KD of 24.6 ± 8.4 μM but with a reduced ligand-to-receptor stoichiometry, consistent with nonproductive domain self-association observed by gel-filtration chromatography; domain II revealed no detectable binding. By contrast, the full-length riboswitch binds a single NAD+ with a KD of 31.5 ± 1.5 μM; dinucleotides NADH and AP2-ribavirin also bind with one-to-one stoichiometry. Unexpectedly, the full-length riboswitch also binds a single ATP equivalent (KD = 11.0 ± 3.5 μM). The affinity trend of the full-length riboswitch is ADP = ATP > NAD+ = AP2-ribavirin > NADH. Although our results support riboswitch sensing of a single NAD+ at concentrations significantly below the intracellular levels of this cofactor, our findings do not support the level of specificity expected for a riboswitch that exclusively senses NAD+. Gene regulatory implications and future challenges are discussed.","journal":"Biochemistry","year":2023,"id":383935,"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":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9581,"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":889288,"name":"Maya E. Blau","orcid":"0000-0002-8948-4982","position":1,"is_corresponding":false},{"id":458608,"name":"Jermaine L. Jenkins","orcid":"0000-0003-2548-3275","position":2,"is_corresponding":false},{"id":467934,"name":"Joseph E. Wedekind","orcid":"0000-0002-4269-4229","position":3,"is_corresponding":false},{"id":1150939,"name":"Yoshita Srivastava","orcid":"0000-0001-9091-9851","position":0,"is_corresponding":true}],"reference_count":71,"raw_metadata":null,"created_at":"2026-07-19T01:17:29.050285Z","pmid":"37947391","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":[]}