The N-terminal intrinsically disordered region of Ncb5or docks with the
cytochrome b5 core to form a helical motif that is of ancient origin
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 L 34MDWIRL 40
motif 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 (S 116QMDWLKLTRT
126) 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.