INTRODUCTION
Ncb5or (NADH cytochrome b5 oxidoreductase, also called Cyb5R4, b5/b5R and b5+b5R) is a cytosolic ferric reductase implicated in diabetes, neurologic diseases and iron homeostasis.1-5Ncb5or is widely expressed in animal cells and localized to endoplasmic reticulum.2, 6 Ncb5or contains two redox domains and one linkage domain that are homologous to cytochrome b5 (Cyb5), cytochrome b5 reductase (Cyb5R), and members of C HORD andS GT1 (CS) family, respectively.6, 7 Ncb5or mediates electron transfer from NADH or NADPH to a redox substrate via FAD and heme cofactors in the b5R and b5 domains, respectively, and proper inter-domain interactions are essential to this process.6Although full-length Ncb5or from human has proven resistant to crystallization to date, we have succeeded in obtaining high-resolution structures of the b5 domain (PDB 3LF5) and a fragment containing the CS and b5R domains (CS/b5R) in complexes with NAD+ and NADP+ (PDB 6MV1 and 6MV2, respectively).8, 9 Despite having the same general fold, the b5 domain exhibits substantially different heme ligation from that of microsomal cytochrome b5 (Cyb5A).8Similarly, the b5R domain differs from microsomal cytochrome b5 reductase (Cyb5R3) in having several multi-residue deletions and insertions that support extensive interactions with the CS domain and reflect a closer relationship to Cyb5R proteins from plants, fungi and protists than to Cyb5R3 from animals.9
The b5 domain of Ncb5or is preceded by a 50-residue N-terminal region that is unique among animal proteins but has homologs in Reduced Lateral Root Formation (RLF) proteins in plants and Increased Recombination Center 21 (IRC21) proteins in fungi. As in Ncb5or, the N-terminal region in the largely unexplored RLF and IRC21 proteins precedes a b5 domain. Herein we report the results of studies aimed at elucidating the structural properties of the N-terminal region of human Ncb5or both in isolation (hereafter N-term), and when natively fused to the b5 domain (hereafter N/b5). Circular dichroism (CD) spectroscopic data revealed that N-term is intrinsically disordered but exhibits significant helical content in N/b5, with an invariant tryptophan residue (Trp37) playing a key role in inducing the helical structure. CD studies further suggested that Trp37, located in a highly conserved L34MDWIRL40 motif in mammalian orthologs, resides in a highly organized environment. While N/b5 constructs representing human Ncb5or failed to form crystals, a recombinant construct of rice RLF comprising residues K101-E218 crystallized readily. The resultant high-resolution X-ray crystallographic structure showed Trp120(corresponding to Trp37 in Ncb5or) to be part of an 11-residue α-helix (S116QMDWLKLTRT126) packing against two of the four helices that surround heme (α2 and α5 in Ncb5or). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues that are equivalent to Ncb5or residues Tyr85 and Tyr88 in α2, Cys124 in α5, and Leu47. In addition to advancing our understanding of Ncb5or structure and drawing our attention to several surface-exposed side chains that may be functionally important, the studies also establish that the N/b5 unit of this multi-domain protein contains a helix motif that is of ancient origin and part of a well-conserved protein structural module.