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

Abstract

PDB references: naturally fused CS and b 5 R domains of human Ncb5or, complex with NAD + , 6mv1; complex with NADP + , 6mv2Supporting information: this article has supporting information at journals.iucr.org/d 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 -sandwich motif of the CS domain. This limits the flexibility between the two domains, which recognize one another via a short -sheet motif and a network of conserved side-chain hydrogen bonds, salt bridges and cation-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. research papers Acta Cryst. (2019). D75, 628-638 Benson et al. CS and cytochrome b 5 reductase domains of Ncb5or 629 research papers Acta Cryst. (2019). D75, 628-638 Benson et al. CS and cytochrome b 5 reductase domains of Ncb5or 637

Characterization and Molecular Mechanism of a Novel Cytochrome b5 Reductase with NAD(P)H Specificity from Mortierella alpina

“…These NCB5Rs, which have the ability to bind NADPH, are formed through genetic mutations, which seriously affect the physiological function of the host . In addition, the structure and sequence alignment showed that, compared with NCB5R3 from animals, the Cyt b 5 R domain of Ncb5or was more closely related to the single-domain NCB5Rs from plants, fungi, and some protists . This study indicates that a natural form of NCB5R accepting NADPH is likely to exist in plants, fungi, and protists.…”

“…At room temperature, 0.2 mL of reaction mixture contains 0.4 mM DCIP, 0.5 μg of pure protein and 100 mM potassium phosphate buffer (pH 7.0). When 0.25, 0.5, 1.0, 2.0, and 4.0 mM NAD­(P)H were added to the mixture, the absorbance changes at 600 nm were recorded every 10 s for 10 min using Thermo Scientific Fluoroskan Ascent, respectively.…”

Cytochrome b5 reductases: Redox regulators of cell homeostasis

“…The N -terminal b5 domain is linked to the C-terminal b5R domain via a CS ( C HORD- S GT1) domain, comprised of roughly 90 amino acid residues and nine β-sheets ( 55 ). The CS domain of CYB5R4 differs from its structural homologs, featuring an additional β-sheet structure involving residues G256 and P267, forming two strands (β8 and β9) separated by a five-residue loop that orient in an antiparallel fashion ( 125 ). Beyond P267, a classic type I β-turn from R268 and T271 forms a linkage to the b5R domain ( 125 ).…”

Human Hsp90 cochaperones: perspectives on tissue-specific expression and identification of cochaperones with similar in vivo functions

“…The reason it may be highly expressed in the brain is unknown, but it may be related to its interaction with Rac, a member of the Ras superfamily (Courilleau et al 2000). NCB5or (NADH-cytochrome b 5 oxidoreductase) is implicated in diabetes and neurological diseases, but the role of Hsp90 in these functions is unclear (Benson et al 2019).…”