Figure 3.

Structure detail of the binding pocket with NAD⁺. (A) Schematic secondary structure of NAD⁺-II riboswitch core architecture. (B) Surface representation of NAD⁺-II riboswitch NAD⁺-binding pocket in Mol A. The NMN moiety of NAD⁺ intercalates into the binding pocket, while the ADP moiety of NAD⁺ points outside. (C) Surface representation of NAD⁺-II riboswitch NAD⁺-binding pocket in Mol B. The NMN moiety of NAD⁺ intercalates the binding pocket, while the ADP moiety of NAD⁺ is stacked by junction J1. (D) NMN moiety of NAD⁺ participates in the formation of a triple with G41 and C54, which stacks between two base platform, G6-U7-C40-A55 and C5-G42-A53. (E, F) NMN moiety of NAD⁺ in both Mol A and Mol B forms extensive hydrogen bond interaction with C54 and G41. The composite omit maps (contoured at 1.0 σ level) of the ligand NAD⁺ in Mol A and Mol B is shown in (E) and (F) respectively. (G) Overlay of ITC titration heat plot of NAD⁺-II riboswitch WT RNA in comparison with mutants concerning nucleotides G41 and C54. (H) Close-up view of the coordination of two Mg²⁺ cations to RNA and ligand NAD⁺ in Mol A: M1 is inner-sphere coordinated with O6 of G42 and the ribose oxygen of NMN moiety of NAD⁺; M2 exhibits one inner-sphere coordination with the phosphate oxygen of NAD⁺, one water-mediated interaction with O6 of G4. (I) Hydrogen bond interactions between ADP moiety of NAD⁺ with A16 and A17 from J1 in Mol B.