The N-terminal intrinsically disordered region of Ncb5or docks with the cytochrome b₅ core to form a helical motif that is of ancient origin

NADH cytochrome b(5) oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b(5) (b(5)) and cytochrome b(5) reductase (b(5)R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b(5) domain and a construct containing the CS and b(5)R domains (CS/b(5)R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved (LMDWIRL40)-M-34 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b(5) domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b(5) domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25-129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp(120) (corresponding to invariant Trp(37) in Ncb5or) to be part of an 11-residue alpha-helix (S(116)QMDWLKLTRT(126)) packing against two of the four helices in the b(5) domain that surround heme (alpha 2 and alpha 5). The Trp(120) side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr(85) and Tyr(88) (alpha 2), Cys(124) (alpha 5), and Leu(47) in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp(37) in nucleating the formation of the N-terminal helix, whose location in the N/b(5) module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b(5) module as reflected by its existence in a class of cytochrome b(5) proteins from three kingdoms among eukaryotes.