ABSTRACT
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) linker domain. Ncb5or redox activity
depends on proper interdomain 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 b5 domain and a construct
containing the CS and b5R domains (CS/b5R). Ncb5or also contains an
N-terminal intrinsically disordered region of 50 residues with a
distinctive, conserved L34MDWIRL40motif that has no homologs in animals but is present in root lateral
formation protein (RLF) in rice and Increased Recombination Center 21
(IRC21) in baker’s yeast, and in these proteins, it is likewise attached
to a b5 domain. After unsuccessful attempts at crystallizing a human
Ncb5or construct comprising the N-terminal region naturally fused to the
b5 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 Trp120 (corresponding to invariant Trp37 in Ncb5or) to be part
of an 11-residue α-helix
(S116QMDWLKLTRT126) packing against
two of the four helices in the b5 domain that surround heme (α2 and α5).
The Trp120 side chain forms a network of interactions with the side
chains of four highly conserved residues corresponding to Tyr85 and
Tyr88 (α2), Cys124 (α5), and Leu47 in Ncb5or. Circular dichroism (CD)
measurements of human Ncb5or fragments further support a key role of
Trp37 in nucleating the formation of the N-terminal helix, whose
location in the N/b5 module suggests a role in regulating the function
of this multidomain redox enzyme. This study revealed for the first time
an ancient origin of a helical motif in the N/b5 module as reflected by
its existence in a class of cytochrome b5 proteins from three kingdoms
among eukaryotes.