biostudies-literatureXu XFundamental Research Funds for the Central UniversitiesAustrian Science Fund FWFAustrian Research Promotion AgencyNational Natural Science Foundation of ChinaNational Key Research and Development Project of ChinaZhejiang Universityoutstanding youth fund of Zhejiang Province54-67https://www.ebi.ac.uk/biostudies/studies/S-EPMC9841397biostudies-literatureUnknown51(1)Riboswitches are conserved non-coding domains in bacterial mRNA with gene regulation function that are essential for maintaining enzyme co-factor metabolism. Recently, the pnuC RNA motif was reported to selectively bind nicotinamide adenine dinucleotide (NAD+), defining a novel class of NAD+ riboswitches (NAD+-II) according to phylogenetic analysis. To reveal the three-dimensional architecture and the ligand-binding mode of this riboswitch, we solved the crystal structure of NAD+-II riboswitch in complex with NAD+. Strikingly and in contrast to class-I riboswitches that form a tight recognition pocket for the adenosine diphosphate (ADP) moiety of NAD+, the class-II riboswitches form a binding pocket for the nicotinamide mononucleotide (NMN) portion of NAD+ and display only unspecific interactions with the adenosine. We support this finding by an additional structure of the class-II RNA in complex with NMN alone. The structures define a novel RNA tertiary fold that was further confirmed by mutational analysis in combination with isothermal titration calorimetry (ITC), and 2-aminopurine-based fluorescence spectroscopic folding studies. Furthermore, we truncated the pnuC RNA motif to a short RNA helical scaffold with binding affinity comparable to the wild-type motif to allude to the potential of engineering the NAD+-II motif for biotechnological applications.Nucleic acids researchStructure-based investigations of the NAD+-II riboswitch.PMC9841397BioNMR 8580173202203991940302P 3169191640104P316912021YFC2300300F8011-B2017QN8101031870810LR19C050003Micura REgger MLi CChen HXu XRen AfalseStructure-based investigations of the NAD+-II riboswitch.Riboswitches are conserved non-coding domains in bacterial mRNA with gene regulation function that are essential for maintaining enzyme co-factor metabolism. Recently, the pnuC RNA motif was reported to selectively bind nicotinamide adenine dinucleotide (NAD+), defining a novel class of NAD+ riboswitches (NAD+-II) according to phylogenetic analysis. To reveal the three-dimensional architecture and the ligand-binding mode of this riboswitch, we solved the crystal structure of NAD+-II riboswitch in complex with NAD+. Strikingly and in contrast to class-I riboswitches that form a tight recognition pocket for the adenosine diphosphate (ADP) moiety of NAD+, the class-II riboswitches form a binding pocket for the nicotinamide mononucleotide (NMN) portion of NAD+ and display only unspecific interactions with the adenosine. We support this finding by an additional structure of the class-II RNA in complex with NMN alone. The structures define a novel RNA tertiary fold that was further confirmed by mutational analysis in combination with isothermal titration calorimetry (ITC), and 2-aminopurine-based fluorescence spectroscopic folding studies. Furthermore, we truncated the pnuC RNA motif to a short RNA helical scaffold with binding affinity comparable to the wild-type motif to allude to the potential of engineering the NAD+-II motif for biotechnological applications.2023-01-01T00:00:00Z2023 Jan2026-05-28T22:53:06.541Z2025-04-06T11:12:11.506ZS-EPMC98413973661078910.1093/nar/gkac1227