Figure 1

Structure of the NAD-NudC complex and recognition of NAD by NudC. (A) NudC-mediated hydrolysis of NAD was detected by high-performance liquid chromatography (HPLC). The elution profiles of the standard samples NMN, AMP and NAD were recorded at 254 nm and are shown in the upper panel. The lower panel shows the elution profiles of NAD processed by wild-type NudC and the E178Q mutant. (B) Overall structure of the NAD-NudC complex in two perpendicular views. The NudC homodimer consists of two symmetric subunits (Mol A in green and Mol B in wheat) and resembles a butterfly with a wingspan of approximately 70 Å and a height of 60 Å. Each subunit binds one NAD molecule. NAD is shown in a yellow ball-and-stick model and is indicated by an arrow. Zinc ions are represented by gray spheres. (C) A ribbon representation of the Mol A subunit. The N-terminal domain (NTD) is colored cyan, the zinc-motif is colored bronze, the C-terminal domain (CTD) is colored green, and the Nudix motif within the CTD is colored red. The numbers under the column depict the boundaries of the motifs and domains in NudC. The labels on the helices or strands were used to trace the NudC sequence. (D) The 2Fo-Fc electron density of NAD, contoured at 1σ and shown in blue, is clearly visible in the cavity of the CTD. (E) Close-up view of the NAD binding site. The residues participating in the interactions with NAD are shown as sticks, with nitrogen in blue and oxygen in red. The hydrogen bonds are represented by red dashed lines. The key residues of the hydrophobic cavity that accommodates the NAD nicotinamide moiety are labeled. (F) The recognition of the NAD nicotinamide moiety by NudC. The residues that mediate the interactions with NAD via hydrogen bonds are labeled. (G) The adenine moiety of NAD is stacked with the phenyl rings of F160 on Mol A and Y124 on Mol B. Two hydrogen bonds buttress the stacking effect. (H) The NAD hydrolysis activity of NudC and the mutants. The activity was measured by HPLC. Each experiment was independently repeated three times; average values and standard deviations are shown. (I) Model representation of formation of the NAD-NudC complex.