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.