Crystal structures of the naturally fused CS and cytochrome b₅ reductase (b₅R) domains of Ncb5or reveal an expanded CS fold, extensive CS–b₅R interactions and productive binding of the NAD(P)⁺ nicotinamide ring

Ncb5or (NADH-cytochrome b₅ oxidoreductase), a cytosolic ferric reductase implicated in diabetes and neurological diseases, comprises three distinct domains, cytochrome b₅ (b₅) and cytochrome b₅ reductase (b₅R) 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 b₅ (Cyb5A) and b₅R (Cyb5R3) system, is of interest. A high-resolution structure of the b₅ 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 b₅R 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 b₅R 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 b₅ 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 b₅R 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 b₅R domain is more closely related to single-domain Cyb5R proteins from plants, fungi and some protists than to Cyb5R3 from animals.