Structure-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.