Structural Characterization of Ncb5or, a Multi‐Domain Ferric Reductase Implicated in Diseases
Authors: Zhu Hao , Benson David R. , Deng Bin , Lovell Scott , Battaile Kevin P. , Cooper Anne , Gao F. Philip , Fenton Aron W. 
Published: April 2020

Ncb5or (NADH cytochrome b5 oxidoreductase) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5) and cytochrome b5 reductase (b5R) domains separated by a CHORD‐Sgt1 (CS) domain, plus a 50‐residue N‐terminal region (N‐term). Ncb5or mediates electron transfer from NADH or NADPH to a redox substrate via FAD and heme cofactors in the b5R and b5 domains, respectively. The inter‐domain interactions are essential to Ncb5or redox function. Although full‐length Ncb5or from human has proven resistant to crystallization to date, we succeeded in obtaining high‐resolution structures of the b5 domain (PDB # 3LF5) and a fragment containing the CS and b5R domains in complex with NAD+ (6MV1) or NADP+ (6MV2). Despite the same general fold, the b5 domain exhibits substantially different heme ligation from that of microsomal cytochrome b5 (Cyb5A). Similarly, the b5R domain differs from microsomal cytochrome b5 reductase (Cyb5R3) in having several multi‐residue deletions and insertions that support extensive interactions with CS domain and reflect a closer relationship to Cyb5R proteins from plants, fungi and protists than to Cyb5R3 from animals. Results from electron transfer analysis using various Ncb5or fragments and mismatched Cyb5A and Cyb5R3 proteins suggest that the presence of N‐term and CS domain facilitate the specific b5‐b5R interaction in Ncb5or. In this study, we aim to characterize the structural impact of the N‐term to the b5 domain. The N‐term of Ncb5or has no homologs in animals and contains a distinctive, conserved L34MDWIRL40 motif. Circular Dichroism (CD) data revealed that the N‐term of human Ncb5or is natively disordered, but exhibits significant helical content when naturally fused with the b5 domain. Signals in the near‐UV region further indicated the presence of a tryptophan side chain in a well‐defined environment, suggesting a role for Trp37 in nucleating N‐term helical structure. This is supported by the CD data of the Trp37Ala mutant in an N‐term/b5 construct. The latter was resistant to crystallization, we therefore sought its homolog from plants, the reduced lateral root formation (RLF) protein that comprises a cytochrome b5 domain preceded by a 130‐residue N‐terminal region. There is a 52% sequence identity (74% similarity) when considering the b5 cores and the preceding 22 residues of the N‐terminal regions. A recombinant rice RLF protein comprising residues 100–218 crystallized readily, and the resultant high‐resolution 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 (α1 and α4). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues (Tyr85 and Tyr88 in α1, Cys124 in α4, and Leu46 in Ncb5or). The location of the N‐term helix suggests a role in mediating electron flow from NAD(P)H to its as yet unidentified redox substrate.

Support or Funding Information

Supported by NIH (P30GM110761, P.I.: R.P. Hanzlik) and KU College of Liberal Arts and Sciences.