Crystal structures of the naturally fused CS and cytochrome b(5) reductase (b(5)R) domains of Ncb5or reveal an expanded CS fold, extensive CS-b(5)R interactions and productive binding of the NAD(P)(+) nicotinamide ring.

Ncb5or (NADH-cytochrome b(5) oxidoreductase), a cytosolic ferric reductase implicated in diabetes and neurological diseases, comprises three distinct domains, cytochrome b(5) (b(5)) and cytochrome b(5) reductase (b(5)R) domains separated by a CHORD-Sgt1 (CS) domain, and a novel 50-residue N-terminal region. Understanding how interdomain interactions in Ncb5or facilitate the shuttling of electrons from NAD(P)H to heme, and how the process compares with the microsomal b(5) (Cyb5A) and b(5)R (Cyb5R3) system, is of interest. A high-resolution structure of the b(5) domain (PDB entry 3lf5) has previously been reported, which exhibits substantial differences in comparison to Cyb5A. The structural characterization of a construct comprising the naturally fused CS and b(5)R domains with bound FAD and NAD(+) (PDB entry 6mv1) or NADP(+) (PDB entry 6mv2) is now reported. The structures reveal that the linker between the CS and b(5)R cores is more ordered than predicted, with much of it extending the β-sandwich motif of the CS domain. This limits the flexibility between the two domains, which recognize one another via a short β-sheet motif and a network of conserved side-chain hydrogen bonds, salt bridges and cation-π interactions. Notable differences in FAD-protein interactions in Ncb5or and Cyb5R3 provide insight into the selectivity for docking of their respective b(5) redox partners. The structures also afford a structural explanation for the unusual ability of Ncb5or to utilize both NADH and NADPH, and represent the first examples of native, fully oxidized b(5)R family members in which the nicotinamide ring of NAD(P)(+) resides in the active site. Finally, the structures, together with sequence alignments, show that the b(5)R domain is more closely related to single-domain Cyb5R proteins from plants, fungi and some protists than to Cyb5R3 from animals.