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\begin{document}

\title{The N-terminal intrinsically disordered region of Ncb5or docks with the
cytochrome b5 core to form a helical motif that is of ancient origin}



\author[1]{Hao Zhu}%
\author[2]{David Benson}%
\author[1]{Bin Deng}%
\author[2]{Maithri M. Kashipathy}%
\author[2]{Scott Lovell}%
\author[3]{Kevin P. Battaile}%
\author[2]{Anne Cooper}%
\author[2]{Philip  Gao}%
\author[1]{Aron W. Fenton}%
\affil[1]{The University of Kansas Medical Center}%
\affil[2]{The University of Kansas}%
\affil[3]{New York Structural Biology Center}%


\vspace{-1em}



  
  \date{May 21, 2024}


\begingroup
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\selectlanguage{english}
\begin{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 \textsuperscript{34}MDWIRL \textsuperscript{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 \selectlanguage{greek}α-\selectlanguage{english}helix (S \textsuperscript{116}QMDWLKLTRT
\textsuperscript{126}) packing against two of the four helices in the b5
domain that surround heme (\selectlanguage{greek}α\selectlanguage{english}2 and \selectlanguage{greek}α\selectlanguage{english}5). The Trp120 side chain forms a
network of interactions with the side chains of four highly conserved
residues corresponding to Tyr85 and Tyr88 (\selectlanguage{greek}α\selectlanguage{english}2), Cys124 (\selectlanguage{greek}α\selectlanguage{english}5), 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.%
\end{abstract}%



\sloppy


\textbf{The N-terminal intrinsically disordered region of Ncb5or docks
with the cytochrome b5 core to form a helical motif that is of ancient
origin}

David R. Benson,\textsuperscript{1} Bin Deng,\textsuperscript{4} Maithri
M. Kashipathy,\textsuperscript{5} Scott Lovell,\textsuperscript{5} Kevin
P. Battaile,\textsuperscript{7} Anne Cooper,\textsuperscript{6} Philip
Gao,\textsuperscript{6} Aron W. Fenton,\textsuperscript{3} Hao
Zhu\textsuperscript{2,3,4}

Department of \textsuperscript{1}Chemistry, University of Kansas,
Lawrence, KS 66045, U.S.A.; Department of \textsuperscript{2}Clinical
Laboratory Sciences, \textsuperscript{3}Biochemistry and Molecular
Biology, \textsuperscript{4}Physical Therapy and Rehabilitation Science,
University of Kansas Medical Center, Kansas City, KS 66160,
U.S.A.;\textsuperscript{5}Protein Structure and X-ray Crystallography
Laboratory, \textsuperscript{6}Protein Production Group, The University
of Kansas, 2034 Becker Drive, Lawrence, KS 66047,
USA;\textsuperscript{7}NYX, New York Structural Biology Center, Upton,
NY, 11973, USA.

TITLE RUNNING HEAD: Intrinsically disordered region of Ncb5or

\textbf{AUTHOR INFORMNATION}

\textbf{Corresponding Authors} :

drb@ku.edu (David R. Benson), hzhu@kumc.edu (Hao Zhu)

David R. Benson, Ph.D., 1567 Irving Hill Road, 1140 Gray-Little Hall,
Lawrence, KS 66045, U.S.A., Phone: 785-864-4090, FAX: 785-864-5396,
E-mail: drb@ku.edu;

Hao Zhu, Ph.D., 3901 Rainbow Blvd., MSN 4048G-Eaton, Kansas City, KS
66160, U.S.A., Phone: 913-588-2989, FAX: 913-588-5222, E-mail:
hzhu@kumc.edu.

\textbf{Funding Sources} :

This work was supported by the National Institute of General Medical
Sciences, National Institutes of Health (P30GM110761; R.P. Hanzlik,
University of Kansas). Additional support came from the University of
Kansas Medical Center School of Health Professions (H.Z.) and University
of Kansas College of Liberal Arts and Sciences (D.R.B.). Use of the
IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by
the companies of the Industrial Macromolecular Crystallography
Association through a contract with Hauptman-Woodward Medical Research
Institute. Use of the Advanced Photon Source was supported by the U.S.
Department of Energy, Office of Science, Office of Basic Energy
Sciences, under Contract No. DE-AC02-06CH11357.

\textbf{ABBREVIATIONS}

CD, circular dichroism; Cyb5A, type A cytochrome b5; Cyb5R3, NADH
cytochrome b5 reductase isoform 3; IRC21, Increased Recombination Center
21; NADH, nicotinamide adenine dinucleotide (reduced); Ncb5or, NADH
cytochrome b5 oxidoreductase; RLF, reduced lateral root formation.

\textbf{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\textsuperscript{34}MDWIRL\textsuperscript{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
\selectlanguage{greek}α-\selectlanguage{english}helix (S\textsuperscript{116}QMDWLKLTRT\textsuperscript{126}) packing
against two of the four helices in the b5 domain that surround heme (\selectlanguage{greek}α\selectlanguage{english}2
and \selectlanguage{greek}α\selectlanguage{english}5). The Trp120 side chain forms a network of interactions with the
side chains of four highly conserved residues corresponding to Tyr85 and
Tyr88 (\selectlanguage{greek}α\selectlanguage{english}2), Cys124 (\selectlanguage{greek}α\selectlanguage{english}5), 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.

\textbf{Keywords}

cytochrome b5, circular dichroism, crystallography, helix motif,
intrinsically disordered region.

\textbf{Supporting Information Available} .

\emph{This includes sequence alignments of Ncb5or in animals (N-terminal
region and cytochrome b5 domain) and of the homologs in plants,
fungi/yeasts, protists and parasites, along with prediction plots of
intrinsically disordered regions in human Ncb5or.}

\textbf{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.\emph{\textsuperscript{1-5}}Ncb5or is widely expressed in
animal cells and localized to endoplasmic
reticulum.\emph{\textsuperscript{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 \emph{C} HORD
and\emph{S} GT1 (CS) family, respectively.\emph{\textsuperscript{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.\emph{\textsuperscript{6}}Although 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).\emph{\textsuperscript{8, 9}} Despite having the same
general fold, the b5 domain exhibits substantially different heme
ligation from that of microsomal cytochrome b5
(Cyb5A).\emph{\textsuperscript{8}}Similarly, 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.\emph{\textsuperscript{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
L\textsuperscript{34}MDWIRL\textsuperscript{40} 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 Trp\textsuperscript{120}(corresponding
to Trp\textsuperscript{37} in Ncb5or) to be part of an 11-residue
\selectlanguage{greek}α-\selectlanguage{english}helix (S\textsuperscript{116}QMDWLKLTRT\textsuperscript{126}) packing
against two of the four helices that surround heme (\selectlanguage{greek}α\selectlanguage{english}2 and \selectlanguage{greek}α\selectlanguage{english}5 in
Ncb5or). The Trp\textsuperscript{120} side chain forms a network of
interactions with the side chains of four highly conserved residues that
are equivalent to Ncb5or residues Tyr\textsuperscript{85} and
Tyr\textsuperscript{88} in \selectlanguage{greek}α\selectlanguage{english}2, Cys\textsuperscript{124} in \selectlanguage{greek}α\selectlanguage{english}5, and
Leu\textsuperscript{47}. 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.

\textbf{MATERIALS AND METHODS}

\textbf{Molecular cloning and site-directed mutagenesis.} Human Ncb5or
contains 521 amino acid residues that comprise the N-terminal region and
the b5, b5R and CS domains as previously
defined.\emph{\textsuperscript{8}} These segments of the protein contain
50, 113, 111, and 247 residues, respectively, that also include linker
sequences in the middle two fragments (\textbf{Figure 1A} ).
Nomenclature of the constructs used herein is as follows: N/b5
(Met\textsuperscript{1} through Lys\textsuperscript{137}); N/b5-\selectlanguage{greek}Δ\selectlanguage{english}21
(Gly\textsuperscript{22} through Lys\textsuperscript{137}); N/b5-\selectlanguage{greek}Δ\selectlanguage{english}34
(Met\textsuperscript{35} through Lys\textsuperscript{137}); b5
(Lys\textsuperscript{51} through Lys\textsuperscript{137}); and N-term
(Met\textsuperscript{1} through Leu\textsuperscript{50}). The cDNAs of
all Ncb5or constructs (except N-term) and rice RLF (residues 101-218,
XP\_015647767) were expressed with no epitope tag (pET19b or pET22b).
The cDNA of N-term was cloned into the pE-SUMOpro Kan vector (Life
Sensors, Malvern, CA), which contains the 6XHis-SUMO gene immediately in
front of the multiple cloning site previously
described.\emph{\textsuperscript{10}}Missense point mutants, N/b5-LMAA
(Leu\textsuperscript{34}Ala/Met\textsuperscript{35}Ala) and N/b5-W37A
(Trp\textsuperscript{37}Ala) (\textbf{Figure 1B} ), were generated using
the QuikChange mutagenesis kit (Strategene, La Jolla, CA). All
mutagenesis primers were synthesized by Integrated DNA Technology
(Coralville, IA). All constructs were confirmed by DNA sequencing (ACGT,
Inc., Wheeling, IL).

\textbf{Protein preparation.} Except for N-term, all constructs of human
Ncb5or and rice RLF were expressed in \emph{E.coli} BL21(DE3) or
BL21(DE3)pLysS or BL21(DE3)pRARE cells and purified as previously
described.\emph{\textsuperscript{8}} In combination with size exclusion
chromatography on a Superdex 75 10/300 GL column, ion-exchange
chromatography was performed for b5 and N/b5-\selectlanguage{greek}Δ\selectlanguage{english}34 on a Q HP column, N/b5
and variants (N/b5-W37A, Nb5-LMAA, and N/b5-\selectlanguage{greek}Δ\selectlanguage{english}21) on a SP HP column, and
rice RLF on both Q and SP HP columns. Full-length Ncb5or and its variant
Ncb5or-\selectlanguage{greek}Δ\selectlanguage{english}50 were prepared as previously
described\emph{\textsuperscript{2}} , except that 0.5 mM IPTG was used
for induction in TB media at 15\textsuperscript{o}C. All purification
steps were performed at 4\textsuperscript{o}C. SDS-PAGE was used to
estimate the purity of each protein used in spectroscopic analyses,
kinetics studies and crystallization screening (all greater than 95\%).
All purified proteins were flash frozen in liquid nitrogen and stored as
aliquots at -80\textsuperscript{o}C until use, except for samples
prepared for crystallization screening which were stored at
4\textsuperscript{o}C. The size of each polypeptide product was
confirmed by mass spectrometry. Expected sizes are: N/b5, N/b5-W37A,
N/b5-LMAA, \textasciitilde{}15.6 kD; N/b5-\selectlanguage{greek}Δ\selectlanguage{english}21 and RLF, \textasciitilde{}
13.6 kD; N/b5-\selectlanguage{greek}Δ\selectlanguage{english}34, 11.9 kD; and Ncb5or-b5, 10.2 kD. The ratios of
A\textsubscript{413}/A\textsubscript{280} for purified heme-containing
constructs were as follows: b5 [?] 4, N/b5 and variants, RLF [?] 3.6, Ncb5or
\textasciitilde{}1.0, and Ncb5or-\selectlanguage{greek}Δ\selectlanguage{english}50 \textasciitilde{}1.1. The FAD
content of Ncb5or-b5R was determined by A\textsubscript{461} and used to
represent enzyme concentrations as previously
described.\emph{\textsuperscript{8}} Final protein yields (mg/L): 8
(b5), 5 (N/b5 and variants), 2 (b5R), [?]1 (Ncb5or, Ncb5or-\selectlanguage{greek}Δ\selectlanguage{english}50, RLF).
Solubility in low-salt buffer: N/b5, RLF \textgreater{} b5
\textgreater{} Ncb5or \textgreater{} Ncb5or-\selectlanguage{greek}Δ\selectlanguage{english}50 \textgreater{}
Ncb5or-b5R. Protease inhibitor (Sigma, St. Louis, MO) was used during
protein preparation to prevent proteolytic cleavage at the b5-CS border
of Ncb5or and Ncb5or-\selectlanguage{greek}Δ\selectlanguage{english}50. A SUMOpro Expression System was used to
prepare N-term.\emph{\textsuperscript{10}}Briefly, a 6XHis-SUMO-N-term
fusion protein was expressed in BL21(DE3) cells and purified by Ni-NTA
affinity resin (Qiagen, Valencia, CA). It was then cleaved by a
6XHis-SUMO protease\emph{\textsuperscript{11}} to release 6XHis-SUMO
tag, both of which were removed by nickel resin. The purity and size of
the N-term peptide were confirmed by SDS-PAGE and mass spectrometry
(data not shown).

\textbf{Spectroscopy.} UV/visible spectra were obtained on a Cary 100
Bio spectrophotometer (Varian). The concentrations of oxidized heme in
all constructs were determined with \selectlanguage{greek}ε\selectlanguage{english}\textsubscript{413} = 130
mM\textsuperscript{-1}cm\textsuperscript{-1} as previously
described.\emph{\textsuperscript{2}}Circular dichroism (CD) analyses
were performed on a JASCO-815 spectropolarimeter using protein
concentrations of 4-10 \selectlanguage{greek}µ\selectlanguage{english}M. Far-UV (190-250 nm) spectra were recorded
using a 1 mm cuvette, whereas a 1 cm cuvette was used for near-UV
(250-350 nm) and visible (350-550 nm) CD spectra. All CD spectra are
reported in units of molar ellipticity ({[}\selectlanguage{english}\selectlanguage{greek}θ{]},
deg\selectlanguage{ngerman}·cm\textsuperscript{2}·dmol\textsuperscript{-1}).

\textbf{RLF crystallization.} A purified RLF construct
(Lys\textsuperscript{101}-Glu\textsuperscript{218}) was concentrated to
44.6 mg/mL in 20 mM Tris pH 8.0 for crystallization screening. All
crystallization experiments were set up using an NT8 drop setting robot
(Formulatrix Inc.) and UVXPO MRC (Molecular Dimensions) sitting drop
vapor diffusion plates at 18\textsuperscript{o}C. 100 nL of protein and
100 nL of crystallization solution were dispensed and equilibrated
against 50 \selectlanguage{greek}\textbf{µ} \selectlanguage{english}L of the latter. Crystals approximately
300-500\selectlanguage{greek}\textbf{µ} \selectlanguage{english}m long that displayed a needle morphology were
observed within one day from the JCSG+ screen (Molecular Dimensions)
condition A6 (20\% (w/v) PEG 1000, 100 mM phosphate-citrate pH 4.2, 200
mM lithium sulfate). A cryoprotectant solution composed of 80\%
crystallization solution and 20\% (w/v) glycerol was dispensed (2
\selectlanguage{greek}\textbf{µ} \selectlanguage{english}L) onto the drop, crystals were harvested with a cryoloop
immediately and stored in liquid nitrogen. X-ray diffraction data were
collected at the Advanced Photon Source beamline 17-ID using a Dectris
Pilatus 6M pixel array detector.

\textbf{Data collection and structure refinement.} Intensities were
integrated using XDS\emph{\textsuperscript{12, 13}} via
Autoproc\emph{\textsuperscript{14}} and the Laue class analysis and data
scaling were performed with Aimless\emph{\textsuperscript{15}} which
indicated that the crystals belong to the 2/\emph{m} Laue class with
possible space groups \emph{P} 2 or \emph{P} 2\textsubscript{1}.
Structure solution was conducted by molecular replacement with
Phaser\emph{\textsuperscript{16}} using the Ncb5or b5-domain structure
(PDB 3LF5\emph{\textsuperscript{8}} ) as the search model. The top
solution was obtained in the space group \emph{P} 2 and contained a
dimer in the asymmetric unit. Initial refinement with
Refmac\emph{\textsuperscript{17}} yielded\emph{R} /\emph{R}
\textsubscript{free} = 42.1\%/42.5\% and the model was improved by
automated building with Arp/wARP.\emph{\textsuperscript{18}}Further
refinement and manual model building were conducted with
Phenix\emph{\textsuperscript{19, 20}} and
Coot\emph{\textsuperscript{21}} respectively. TLS parameters
\emph{\textsuperscript{22, 23}} were refined in the later stages to
model anisotropic atomic displacement parameters. Disordered side chains
were truncated to the point for which electron density could be
observed. Structure validation was conducted with
Molprobity\emph{\textsuperscript{24}} and figures were prepared using
the CCP4MG package\emph{\textsuperscript{25}} . Polder omit electron
density maps were calculated using
Phenix.\emph{\textsuperscript{26}}Structure superposition was carried
out with GESAMT.\emph{\textsuperscript{27}}Crystallographic data are
described in \textbf{Table S1} , and the best data set was at 1.55 A
resolution.

\textbf{RESULTS AND DISCUSSION}

\textbf{The N-terminal region of Ncb5or is predicted to be intrinsically
disordered.}

The amino acid sequence of the N-terminal region of human Ncb5or is
shown in \textbf{Figure 1B} , and an alignment of this 50-residue
polypeptide with all known Ncb5or examples in animals is included
in\textbf{Figure S1} . The alignment revealed an overall homology, which
is weaker in the first 21 residues than the subsequent 29-residue
stretch which is rich in Arg and Lys residues and contains five
invariant residues (Lys25, Leu28, Gly31, Trp37 and Gly49). Trp37 is part
of a highly conserved motif that has the sequence
L\textsuperscript{34}MDWIRL\textsuperscript{40} in mammalian orthologs.

The PONDR VL-XT program\emph{\textsuperscript{28-30}}predicts that the
N-terminal region of Ncb5or is intrinsically disordered, in sharp
contrast to the b5, b5R and CS domains (\textbf{Figure S2A} ). Secondary
structure prediction algorithms generally gave inconsistent predictions
for the N-terminal region, but several of them indicated a preference
for \selectlanguage{greek}α-\selectlanguage{english}helical structure for the
L\textsuperscript{34}MDWIRL\textsuperscript{40} sequence. This included
PROF\emph{\textsuperscript{31}} and PSIPRED\emph{\textsuperscript{32,
33}} (\textbf{Figure S2B} ), as well as
SPINE-X\emph{\textsuperscript{34}} (not shown). The latter two \emph{ab
initio} algorithms (\emph{i.e.} , those not utilizing homology modeling)
have been shown to yield the most accurate
predictions.\emph{\textsuperscript{35}}AGADIR\emph{\textsuperscript{36}}
, an algorithm that predicts helical content of peptides on the basis of
helix/coil transition theory, also predicts some helical tendency for
the L\textsuperscript{34}MDWIRL\textsuperscript{40} sequence, albeit
with a population of less than 10\% at pH 7 and 25\selectlanguage{ngerman}°C (\textbf{Figure
S2C} ).

\textbf{Interactions with the b5 domain induce helical structure in the
N-terminal region.}

To compare intrinsic structural properties of the N-terminal region of
human Ncb5or with those it exhibits in the native protein, we performed
CD spectroscopy studies of the 50-residue N-terminal polypeptide
(hereafter N-term) and protein constructs representing the b5 domain
with and without the N-terminal region attached (hereafter N/b5 and b5,
respectively). Far-UV CD spectra of b5 and N/b5 are shown
in\textbf{Figure 2A} (summarized in \textbf{Table 1)} and presented in
molar ellipticity units to better reflect differences in total secondary
structure content. The N/b5 spectrum differs from that of b5 in
exhibiting enhanced negative ellipticity in the vicinity of 200 nm. The
dominant feature in far-UV CD spectra of unstructured (random coil)
polypeptides is a broad band of negative ellipticity centered near 200
nm.\emph{\textsuperscript{37}} The data in \textbf{Figure 2A} therefore
suggests that N/b5 differs from b5 in having a larger number of
disordered residues. This was confirmed by subtracting the b5 spectrum
from the N/b5 spectrum to give the difference spectrum shown in
\textbf{Figure 2C} . However, this difference spectrum also exhibits
positive ellipticity near 190 nm and negative ellipticity in the
vicinity of 220 nm, both suggestive of the presence of helical
structure.\emph{\textsuperscript{38}} In contrast, the corresponding
N-term spectrum (\textbf{Figure 2C} ) is dominated by a negative band
centered at 200 nm, revealing it to be largely unstructured. These
results strongly suggest that the N-terminal region of Ncb5or is
intrinsically disordered, consistent with the predictions described
above, but that it adopts some helical structure when connected to the
b5 domain.

\textbf{The Trp37 side chain resides in a well-structured environment.}

When located in highly asymmetric (\emph{i.e.,} well-structured)
environments, side chains of aromatic residues exhibit CD signals in
overlapping regions of the near-UV (Phe, 250-270 nm; Tyr, 270-290 nm;
Trp, 280-300 nm).\emph{\textsuperscript{38}} As shown in \textbf{Figure
2B} , b5 displays signals in the near-UV region (250- 350 nm)
attributable to the fact that it contains 2 Phe, 4 Tyr, and 2 Trp
residues. Comparatively stronger signals, with significant fine
structure, appear in the near-UV CD spectrum of N/b5 (\textbf{Figure 2B}
). The N-terminal region contains only two aromatic residues, Phe9 and
Trp37. Given that the strongest feature in the near-UV CD signature of
N/b5 appears at 286 nm, we conclude that the major contributor is Trp37,
which is part of the invariant mammalian Ncb5or
L\textsuperscript{34}MDWIRL\textsuperscript{40} sequence noted above. In
contrast, the corresponding spectrum of N-term exhibits no such signal
at 286 nm (\textbf{Figure 2D} ).

Aromatic residues in well-ordered regions of proteins often contribute
to far-UV CD spectra as well. In the case of Trp, positive bands in the
225-250 nm region are a particularly common
feature.\emph{\textsuperscript{39, 40}} Trp37 is therefore a likely
source of the positive feature in the difference CD spectrum of N-term
centered at 230 nm (\textbf{Figure 2C} ), another conjecture which is
supported by additional CD data described below. Notably, N-term
exhibited no signals in the near-UV region (\textbf{Figure 2D} ) and
displays negative ellipticity in the vicinity of 230 nm (\textbf{Figure
2C} ). The results described in this section allow us to conclude that
interactions between the N-terminal region and the b5 domain induce
secondary structure in the former, and that this structural transition
is accompanied by adoption of tertiary structure involving Trp37. This
conclusion was further tested with truncation and point mutants of N/b5.

\textbf{Trp37 plays a key role in the interactions between the
N-terminal region and the b5 domain.}

We generated two truncation mutants of N/b5 (\textbf{Figure 1} ) and
examined the effects of the truncations on CD spectra. The first mutant
involved the removal of residues 1-21, the portion carrying the lowest
conservation among animals, to yield N/b5-\selectlanguage{greek}Δ\selectlanguage{english}21. Residues 1-34 were also
removed to produce a domain fragment (N/b5-\selectlanguage{greek}Δ\selectlanguage{english}34) analogous to the
truncated Ncb5or that was initially cloned.\emph{\textsuperscript{6}}

Deleting the first 21 residues of N/b5 had no significant effect on the
near-UV CD spectrum, or on the intensity of the positive far-UV CD band
near 230 nm (\textbf{Figures 2E-F, Table 1} ). The most notable effect
on CD spectra caused by removing residues 1-21 was a decrease in
negative ellipticity of the lowest wavelength far-UV band, along with a
slight red shift of that band. These observations suggest that residues
1-21 are almost exclusively disordered and show that they play no role
in the secondary and tertiary structure formation resulting from the
interactions between the N-terminal region and the b5 core. They also
support our conclusion that the near-UV CD signals and the positive
far-UV feature near 230 nm arise from Trp37, with no contribution from
Phe9.

Comparison of the far-UV CD difference spectra for N/b5-\selectlanguage{greek}Δ\selectlanguage{english}21 and N/b5-\selectlanguage{greek}Δ\selectlanguage{english}34
(\textbf{Figure 2E, Table 1} ) shows that deleting the additional
residues 22-34 decreased, but did not abolish, secondary structure
content. In addition, \textbf{Figure 2F} (also \textbf{Table 1} )
reveals diminished intensity for the near-UV CD signals attributable to
Trp37 in N/b5-\selectlanguage{greek}Δ\selectlanguage{english}34, but the signals in this region are much more like
those in the spectrum of N/b5 than of b5. This suggests that Trp37 is
involved in tertiary structure in both truncation mutants, but that
removal of residues 22-34 causes subtle disruption of that tertiary
structure.

The evidence obtained with the truncation mutants strongly indicates a
key role for Trp37 in interactions between the N-terminal region and the
b5 domain of Ncb5or. We therefore examined the effect of mutating Trp37
to Ala on CD spectra of N/b5. Subtracting the far-UV CD spectrum of b5
from that of N/b5-W37A yielded a difference spectrum similar to the
spectrum of N-term (\textbf{Figure 2G} ). Moreover, the W37A mutation in
N/b5 resulted in loss of the near-UV CD features present in the N/b5
spectrum (\textbf{Figure 2H} ). It can therefore be concluded that the
W37A mutation in N/b5 abolishes both secondary and tertiary structure.
In comparison, the difference spectrum obtained by subtracting the
far-UV CD spectrum of N/b5-LMAA from that of N/b5 exhibits modestly
increased intensity of the negative band near 220 nm (\textbf{Figure 2G,
Table 1} ) and of the positive band near 190, suggesting a small
increase in helix content. This may be attributable to the fact that Ala
has a much higher helix propensity than do Leu and
Met.\emph{\textsuperscript{41}} More notably, the LM-AA mutation does
not significantly alter the shape or intensity of the near-UV and far-UV
CD features attributable to Trp37 (\textbf{Figure 2G-H} ). However, the
CD Soret band signal for the LMAA mutant is virtually identical to that
of Ncb5or-b5 (\textbf{Figure 2H} ). These observations suggest that the
LM-AA mutation does not alter the ability of N-term to dock with the b5
core, with associated adoption of tertiary structure involving Trp37,
but that it alters (or perhaps weakens) interactions between the
N-terminal region and the b5 core.

The results described in this section indicate that residues spanning
Met35 through Leu50 are sufficient to induce secondary structure (likely
helical) and accompanying tertiary structure in the N-terminal region
when it docks with the b5 core. The results also show that Trp37 plays
an essential role in inducing this structural transition, suggesting a
specific recognition site on the b5 domain surface. These results
further indicate that the induced secondary structure is propagated
toward the N-terminus but does not extend beyond Gly22. This additional
secondary structure appears to stabilize the tertiary structure.

\textbf{Plant and fungal proteins identified as potential models for
N/b5}

Efforts to confirm the results of the CD studies described above via
X-ray crystallographic studies of the N/b5 constructs were unfortunately
not successful, prompting us to seek potential homologs that might
crystallize more readily. BLAST searches using human Ncb5or residues
1-50 as the query revealed only a few other proteins that contain a
homologous polypeptide, all from plants and fungi. In all cases the
homologous polypeptide precedes a b5 domain having significant homology
to that of Ncb5or and contains a Trp residue analogous to Trp37 of
Ncb5or but no additional domains or regions (\textbf{Figures S1, S3,
S4)} . No structural characterization has been performed for these
homologues, and only two have been characterized functionally: RLF
(\emph{R} educed \emph{L} ateral root \emph{F} ormation protein) from
plants (e.g., rice, \emph{Arabidopsis} and glycine) and IRC21 (\emph{I}
ncreased \emph{R} ecombination \emph{C} enter 21) protein from yeast
(e.g., \emph{Saccharomyces cerevisiae} and \emph{S. pombe} ). RLF
appears to regulate lateral root formation independently of
ARF7/19-mediated auxin signaling\emph{\textsuperscript{42}} , whereas
IRC21 functions in chromatin remodeling\emph{\textsuperscript{43}} and
DNA repair\emph{\textsuperscript{43-45}}likely through its binding to
lipid and protein phosphatase.\emph{\textsuperscript{46, 47}}

The N-terminal regions of plant RLF proteins are approximately
130-residues in length (\textbf{Figure 3A-B)} and contain a sequence
spanning residues 115-121
(Q\textsuperscript{115}MDWLKL\textsuperscript{121}) that is strikingly
similar to residues 34-40 of Ncb5or which encompass Trp37
(L\textsuperscript{34}MDWIRL\textsuperscript{40}). The N-terminal
regions of IRC21 proteins are approximately 77 residues in length, and
in that respect are more like Ncb5or than are the plant RLF proteins.
However, there is weaker homology for residues 61-67
(A\textsuperscript{61}LDWHSL\textsuperscript{67}) that correspond to
residues 34-40 of Ncb5or (\textbf{Figure 3A-B} ). In addition, the
sequence separating this signature from the b5 core is approximately 40
residues longer in IRC21 proteins than in Ncb5or and RLF, an insertion
that may have arisen subsequent to divergence of the plant, fungi and
animal kingdoms.\emph{\textsuperscript{48}}Yeast IRC21 and rice RLF were
used for these comparisons (\textbf{Figures 3B} ). For these reasons we
selected rice RLF protein for our structural studies, specifically
residues 101-218, corresponding to residues 22-137 of human Ncb5or.

\textbf{The b5 cores of Ncb5or and the rice RLF protein have identical
folds.}

The rice RLF construct crystallized readily as a non-crystallographic
dimer and could be modeled from
Phe\textsuperscript{111}-Glu\textsuperscript{218} in subunit A and
Lys\textsuperscript{113}-Phe\textsuperscript{213} in subunit B. The fold
comprises six \selectlanguage{greek}α-\selectlanguage{english}helices and four \selectlanguage{greek}β-\selectlanguage{english}sheets (\textbf{Figure 4A-B} ) that
are arranged as follows: \selectlanguage{greek}α\selectlanguage{english}1
(Ser\textsuperscript{116}-Thr\textsuperscript{126}), \selectlanguage{greek}β\selectlanguage{english}1
(Arg\textsuperscript{140}-Ile\textsuperscript{142}), \selectlanguage{greek}α\selectlanguage{english}2
(Leu\textsuperscript{144}-Lys\textsuperscript{148}), \selectlanguage{greek}β\selectlanguage{english}2
(Trp\textsuperscript{157}-Leu\textsuperscript{160}), \selectlanguage{greek}β\selectlanguage{english}3
(Arg\textsuperscript{163}-Asn\textsuperscript{166}), \selectlanguage{greek}α\selectlanguage{english}3
(Ala\textsuperscript{168}-Phe\textsuperscript{173}), \selectlanguage{greek}α\selectlanguage{english}4
(Val\textsuperscript{178}-Met\textsuperscript{182}), \selectlanguage{greek}α\selectlanguage{english}5
(Thr\textsuperscript{190}-His\textsuperscript{197}), \selectlanguage{greek}α\selectlanguage{english}6
(Phe\textsuperscript{202}-Leu\textsuperscript{205}), \selectlanguage{greek}β\selectlanguage{english}4
(Leu\textsuperscript{210}-Leu\textsuperscript{214}). Note that the
numbering is based on the NCBI reference sequence XP\_015647767.1. The
b5 core of RLF is identical to that of the Ncb5or b5 domain, with
superposition yielding an RMSD deviation of 1.01 \selectlanguage{ngerman}Å (80 residues) between
C\selectlanguage{greek}α \selectlanguage{english}atoms (\textbf{Figure 5} ).

The heme molecules are positioned within a cleft formed by helices
\selectlanguage{greek}α\selectlanguage{english}3-\selectlanguage{greek}α\selectlanguage{english}6. It is important to note that the corresponding helices in the
published structure of the Ncb5or b5 domain, and in structures of single
domain b5 family members, are numbered \selectlanguage{greek}α\selectlanguage{english}2-\selectlanguage{greek}α\selectlanguage{english}5. In the comparison of the
b5 and N/b5 structures we will refer to the relevant helices exclusively
as \selectlanguage{greek}α\selectlanguage{english}3-\selectlanguage{greek}α\selectlanguage{english}6. Like all cytochrome b5 family members, the heme iron is
coordinated by the side chains of two histidine residues (His174 and
His197). His174 (His89 in Ncb5or) is located in the loop separating \selectlanguage{greek}α\selectlanguage{english}3
and \selectlanguage{greek}α\selectlanguage{english}4, while His197 (His112 in Ncb5or) constitutes the C-terminal
residue in helix \selectlanguage{greek}α\selectlanguage{english}5. As observed for His89 and His112 in the crystal
structure of the Ncb5or b5 domain, the imidazolyl moieties of His174 and
His197 in the rice RLF structure are nearly orthogonal to one another
(\textbf{Figure 6B} ). The angle between the mean planes defined by the
indole rings is 77.6\textsuperscript{o} (subunit A) and
77.2\textsuperscript{o} (subunit B) as calculated using
Mercury.\emph{\textsuperscript{49}} The corresponding angles for His89
and His112 in the two subunits of the Ncb5or b5 domain structure (3LF5)
are 83.2\selectlanguage{ngerman}° and 81.3°.\emph{\textsuperscript{8}} This structural feature
distinguishes these proteins (and likely IRC21 as well), from the better
known microsomal and mitochondrial isoforms of cytochrome b5, in which
angles between the His ligand side chains are closer to
20°.\emph{\textsuperscript{8}}

We reported that the b5 core of Ncb5or differs from the better known
microsomal and mitochondrial isoforms of cytochrome b5 in having a
strictly conserved tryptophan residue (Trp114) at the mouth of the heme
binding pocket, two residues removed from heme ligand His112. The
intervening residue, Arg113, is invariant among mammalian Ncb5or
isoforms. In the crystal structure of the b5 domain of Ncb5or the side
chain of Trp114 is located on the protein exterior and is substantially
solvent-exposed (\textbf{Figure 6A} ), with its only inter-protein
interactions involving the side chain of adjacent residue Arg113. The
rice RLF residues corresponding to the Ncb5or
His\textsuperscript{112}ArgTrp\textsuperscript{114} sequence (His197,
Ala198, and Trp199) are strictly conserved among known members of this
protein family. In the RLF crystal structure the Trp199 side chain
projects into solvent, but as shown in \textbf{Figure 6A} its side chain
conformation differs from that of Trp114 in the Ncb5or b5 domain
structure, enabling hydrogen bonding between its N\selectlanguage{greek}ε-\selectlanguage{english}H and one of the
heme propionate groups. Manually changing the side chain torsional
angles of Trp114 in the Ncb5or b5 domain crystal structure (\selectlanguage{greek}χ\selectlanguage{ngerman}º = -53.9;
\selectlanguage{greek}χ\selectlanguage{english}2 = 115.7\selectlanguage{ngerman}º) to match those of Trp199 in the new rice RLF structure (\selectlanguage{greek}χ\selectlanguage{ngerman}º
= 58.9; \selectlanguage{greek}χ\selectlanguage{english}2 = 85.4\selectlanguage{ngerman}º), using PyMol, showed that Trp114 could form an
analogous hydrogen bond with heme without introducing unfavorable steric
interactions. Given that this solvent exposed Trp residue is strictly
conserved among all known Ncb5or and RLF proteins (and in IRC21 proteins
as well), its ability to form a hydrogen bond with heme suggests that it
serves an essential functional role.

Yet another distinguishing feature shared by the b5 cores of Ncb5or and
rice RLF is an irregular helix (\selectlanguage{greek}α\selectlanguage{english}6) in the heme-binding pocket,
featuring a central kink that leaves Leu205 (Met120 in Ncb5or) without
an intra-helical hydrogen bond. As will be noted in the following
section, this kink plays an important role in interactions with the
N-terminal region.

While carrying out the studies reported herein, we became aware of
reports of b5 family members in \emph{Giardia} and some other parasitic
organisms with strong homology to the b5 domains of Ncb5or, RLF and
IRC21 proteins. As shown by some representative examples
in\textbf{Figure S5} , these proteins feature an N-terminal region,
albeit generally shorter than those in Ncb5or, RLF and IRC21 and without
a Trp residue analogous to Trp37 in Ncb5or.\emph{\textsuperscript{50}}
Many of them have also maintained a surface tryptophan residue analogous
to Trp114 of Ncb5or. The \emph{Giardia} proteins were shown to exhibit
redox potentials\emph{\textsuperscript{51}} similar to those determined
for Ncb5or,\emph{\textsuperscript{2}} which are considerably more
negative than for microsomal b5s. The\emph{Giardia} proteins and Ncb5or
also share EPR spectroscopic signatures that are characteristic of
orthogonal His ligands.\emph{\textsuperscript{51}} It is reasonable to
conclude that these proteins have evolved from the common N/b5 ancestor
of Ncb5or, RLF, and IRC21, perhaps with divergent
functions.\emph{\textsuperscript{50, 51}} This notion is supported by
the observation of cytochrome b5 proteins in protists
(\emph{Trypanosoma}and \emph{Dictyostelium)} that share the same b5
core\emph{\textsuperscript{50}} and the N-terminal motif as that in
Ncb5or, including the Trp114 and Trp37 residues, respectively
(\textbf{Figure S5} ).

\textbf{The rice RLF structure confirms N-terminal helix formation
involving the Trp120 side chain.}

Trp120 in RLF (corresponding to Trp37 in Ncb5or) is part of an
11-residue \selectlanguage{greek}α-\selectlanguage{english}helix, designated \selectlanguage{greek}α\selectlanguage{english}1
(S\textsuperscript{116}QMDWLKLTRT\textsuperscript{126}), that packs
against the side of the heme-binding pocket defined by helices \selectlanguage{greek}α\selectlanguage{english}3 and \selectlanguage{greek}α\selectlanguage{english}6
(\textbf{Figure 7} ). In this and subsequent sections, all corresponding
residues in Ncb5or are listed in parentheses. A 17-residue stretch
connects \selectlanguage{greek}α\selectlanguage{english}1 to \selectlanguage{greek}α\selectlanguage{english}2, the first element of defined secondary structure in
the ``classic'' b5 core (\selectlanguage{greek}α\selectlanguage{english}2 is the only helix in the b5 core that does
not comprise part of the heme binding pocket). The corresponding stretch
in Ncb5or is two residues shorter, with the gap appearing close to the
junction with \selectlanguage{greek}α\selectlanguage{english}2. There are multiple conserved residues in the RLF and
Ncb5or loops, and in the RLF structure several of them exhibit specific
backbone and side chain interactions both within the loop and with the
b5 core. These include two three-residue turns featuring a single
backbone hydrogen bond, the first between Asp129 and Gly132 (Asp46 and
Gly49), and the second between Leu133 and Gln136 (Leu 50 and Arg53). The
Asp129 (Asp46) side chain in the first turn extends this motif via
hydrogen bonds to the backbone amide N-H groups of Leu133 and Lys134
(Leu50 and Lys51), residues which can be considered to represent the
transition between N-term and the b5 core. The side chain of Leu133
(Leu50) is buried and engages in hydrophobic interactions with several
nearby residues, namely C\selectlanguage{greek}β \selectlanguage{english}of Ala131 (Thr48), C\selectlanguage{greek}β \selectlanguage{english}of Gln136 (Arg53), and
C\selectlanguage{greek}α \selectlanguage{english}of Asn138 (Ile55). It also packs against the side chain of Val211
(Val126) which is in strand \selectlanguage{greek}β\selectlanguage{english}4 of the b5 core. All the residues in this
hydrogen bonded array other than solvent exposed Gln136 (Arg53) are
conserved in RLF and Ncb5or proteins. Likely because of these
interactions, this polypeptide segment is well-ordered. Packing between
the N-terminal helix and the b5 core involves primarily hydrophobic
interactions and does not noticeably impact the b5 core fold. Consistent
with the results of far-UV CD studies described above, the Trp120 side
chain is situated in an environment with well-defined tertiary structure
that includes hydrophobic packing interactions with the side chains of
four residues in \selectlanguage{greek}α\selectlanguage{english}3 and \selectlanguage{greek}α\selectlanguage{english}6, each corresponding to a residue that is
highly conserved in Ncb5or: Tyr170 and Phe173 in \selectlanguage{greek}α\selectlanguage{english}3 (Tyr85 and Tyr88);
and Leu206 and Cys209 in \selectlanguage{greek}α\selectlanguage{english}6 (Leu121 and Cys124). The Trp120 side chain
engages in additional hydrophobic interactions with the nearby side
chains of Leu121 and Thr124 in \selectlanguage{greek}α\selectlanguage{english}1 (Ile38 and Thr41) and with the side
chain of Leu130 (Leu47), located in the first 3-residue turn in the
17-residue loop connecting \selectlanguage{greek}α\selectlanguage{english}1 to \selectlanguage{greek}α\selectlanguage{english}2.

It is worth noting that the interaction between the Trp120 and Cys209
side chains (Trp37 and Cys124) involves the cysteine thiol (SH group)
packing against the tryptophan pi system. Such interactions are
relatively common and are thought to play a stabilizing
role.\emph{\textsuperscript{52, 53}} Both residues are invariant in
Ncb5or proteins.

The Trp120 (Trp37) side chain has one additional contact, a hydrogen
bond between its N-H group and the backbone carbonyl of Leu205 in \selectlanguage{greek}α\selectlanguage{english}6
(Met120). As noted in the previous section, the backbone carbonyl of
that residue does not form an intra-helix H-bond due to a kink in \selectlanguage{greek}α\selectlanguage{english}6.
The present results suggest that the \selectlanguage{greek}α\selectlanguage{english}6 kink may have evolved to serve a
distinct functional role in these proteins.

The only other polar contacts between \selectlanguage{greek}α\selectlanguage{english}1 and the b5 core comprise a
network of hydrogen bonds involving the side chains of the first two \selectlanguage{greek}α\selectlanguage{english}1
residues Ser116 and Gln117 (Ser31 and Leu32), the phenolic side chain of
Tyr170 in \selectlanguage{greek}α\selectlanguage{english}3 (Tyr85), and the backbone carbonyl of Phe173 in \selectlanguage{greek}α\selectlanguage{english}3 (Tyr88).
While this network of hydrogen bonds may be important in stabilizing \selectlanguage{greek}α\selectlanguage{english}1,
it is noteworthy that the polar OH group of the Tyr170 side chain also
makes van der Waals contact with one of the heme \emph{meso} positions.
This counterintuitive positioning of a polar group suggests a potential
functional role. An intriguing possibility is that oxidation of ferrous
heme to ferric heme during reduction of the downstream substrate is
accompanied by deprotonation of the phenolic group. This could
conceivably facilitate a coupled electron/proton transfer process during
substrate reduction. Alternatively, it could allow for electrostatic
stabilization of the b5 domain when heme is in the ferric oxidation
state and bears a formal positive charge.

\textbf{Several conserved residues in the N-terminal helix have
solvent-exposed side chains.}

The side chains of several other highly conserved residues in the
N-terminal helix project into solvent (\textbf{Figure 8} ), including
three in the vicinity of Trp120: Met118, Asp119, and Lys122
(corresponding to Met35, Asp36, and Arg39 in Ncb5or). Particularly
notable in this group is Met118 (Met35). The thioether side chain of
methionine is considered nonpolar, and indeed methionine is often
subject to evolutionarily conservative replacement by residues with
aliphatic side chains via single codon mutations (leucine, isoleucine,
and valine). Only a few examples of such a mutation, specifically Met to
Leu, exist among known Ncb5or proteins, and only in non-mammalian
organisms (\textbf{Figure S1} ). Methionine differs from the aliphatic
amino acids it often replaces in that its thioether functional group can
serve functional roles (for example, as a ligand to heme iron in
cytochrome \emph{c} ). The highly conserved nature of Met35 in Ncb5or,
considered in the context of its solvent-exposed location, is suggestive
of an essential functional role.

\textbf{CONCLUSION}

This study represents the first \emph{in vitro} characterization of the
N-terminal region of Ncb5or, a polypeptide segment not found in any
other animal proteins, but which shares a common ancestor with RLF
proteins in plants and IRC21 proteins in fungi. The N-terminal regions
in all three of these protein families precede highly homologous
cytochrome b5 domains, and share sequence similarities that suggest that
the results we have obtained using N/b5 constructs derived from human
Ncb5or and rice RLF protein will pertain to the IRC21 proteins as well.
Specifically, we have shown that the N-terminal region is intrinsically
disordered but that a 11-residue motif adopts a helical conformation
when natively attached to the b5 core, a structural transition that is
nucleated by a strictly conserved tryptophane residue (Trp37 in human
Ncb5or). Such docking-induced folding is a common theme for
intrinsically disordered regions of mammalian proteins that are often
involved in DNA or RNA binding, substrate binding, and protein-protein
interactions\emph{\textsuperscript{54-57}} , of which the specificity is
generally mediated by motif structures.\emph{\textsuperscript{58, 59}}
In this context, it is worth investigating whether IRC21 utilizes the
helical motif in the Nb5 module to activate protein phosphatase 2A for
DNA damage response in yeasts.\emph{\textsuperscript{47}}Intrinsically
disordered regions appear to be enriched in disease-related proteins,
such as the N-terminal transactivation domain of tumor suppressor
p53,\emph{\textsuperscript{60}}alpha-synuclein in Parkinson's
disease\emph{\textsuperscript{61}} , tau protein in Alzheimer's
disease\emph{\textsuperscript{62}} , and in a number of proteins
associated with cardiovascular
disease.\emph{\textsuperscript{63}}Mutation of the intrinsically
disordered region of carboxyl ester lipase in pancreatic acinar cells
can cause lean diabetes, likely as a result of protein
mis-folding.\emph{\textsuperscript{64}}Functional contribution of the
N-terminal intrinsically disordered region and the helical motif to
Ncb5or's cellular function and related disease pathways is being
investigated.

The presence of the helical motif within the N/b5 module in three
kingdoms of eukaryotes that diverged about 1.6 billion years
ago\emph{\textsuperscript{48, 65}} suggests an ancient heritage of
Ncb5or. This is consistent with our previous observation of closer
similarities of its b5R domain to monomeric b5R proteins from plants,
fungi and protists than to mammalian Cyb5R3
proteins.\emph{\textsuperscript{9}} The nucleated N-terminal helix lies
between two of the four helices that comprise the heme binding pocket
and is connected to what we have previously referred to as the b5 core
by a well-structured polypeptide. The N-terminal region in Ncb5or and
the plant and fungal proteins could conceivably comprise an integral
part of their b5 cores, which would thus be expanded relative to other
modern cytochrome b5 superfamily members. Alternatively, the N-terminal
regions of these proteins could function to regulate their enzymatic
functions, through reversible docking. Suggestive of this latter
scenario is the observation that the 17 residues separating the
N-terminal helix \selectlanguage{greek}α\selectlanguage{english}1 from \selectlanguage{greek}α\selectlanguage{english}2 in rice RLF protein exhibit few interactions
with the ``classic'' portion of the b5 core, in contrast to the
17-residue helix that is nucleated by the Trp37-dependent docking
interactions. These possibilities will be the subject of future studies.
A low homology in the N-terminal region of cytochrome b5 proteins of
parasites, including the absence of the helical motif, is likely a
result of diminishing selection pressure for the above-mentioned
motif-mediated function due to the unique parasite-host interaction and
pathophysiology.

Future studies will also be aimed at probing potential functional roles
of other strictly conserved residues in Ncb5or emphasized above. These
include Met35 in the N-terminal helix, and Trp114 which is located two
residues from heme ligand His112. The side chains of both these residues
project conspicuously into solvent, locations that would subject them to
random mutation were they not essential for protein function, especially
since they are encoded by a single
codon.\emph{\textsuperscript{66}}Functional roles could include
modulating heme reduction by the b5R domain or reduction by heme of the
still unknown substrate.

\textbf{ACKNOWLEDGMENT}

We thank Christopher D. Lima (Sloan-Kettering Institute, New York) for
the SUMO protease as a gift.

\textbf{STRUCTURE DEPOSITION}

Coordinates and structure factors for the root lateral formation protein
(RLF) b5-domain structure were deposited to the Worldwide Protein
Databank (wwPDB) with the accession code 8TGB.

\textbf{Table 1} . Signature signals of CD spectra for N/b5, its
variants, and b5 of human Ncb5or.\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Molar ellipticity (deg*cm2/dmol)\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Far-UV (minus b5)\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Far-UV (minus b5)\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Helix\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Near-UV\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Tertiary structure\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Soret\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Heme contact\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
190 nm\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
220 nm\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
286 nm\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
413 nm\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
N/b5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
544543\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-310424\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Yes\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-35041\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Yes\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-82501\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Yes\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
N/b5-\selectlanguage{greek}Δ\selectlanguage{english}21\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
778363\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-312167\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Yes\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-36453\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Yes\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-84650\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Yes\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
N/b5-LMAA\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
764345\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-396791\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Yes\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-34688\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Yes\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-69546\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
No\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
N/b5-\selectlanguage{greek}Δ\selectlanguage{english}34\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
636442\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-136573\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Yes\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-27727\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Partial\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-70516\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
No\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
N/b5-W37A\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-132660\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-245259\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
No\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-346\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
No\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-72577\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
No\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
b5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
N/A\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
2262\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
No\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-71620\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
No\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Molar ellipticity values of far-UV signals (190 and 220 nm) are obtained
from difference CD spectra (\textbf{Figures 2C,E,G} ) and indicate
secondary structure in the N-terminal domain. Its tertiary structure and
impact on the heme center are suggested by molar ellipticity values of
near-UV (286 nm) and Soret (413 nm) signals, respectively, from CD
spectra (\textbf{Figures 2B,F,H} ). All Molar ellipticity values are
presented in deg*cm2/dmol. N/A: not applicable. \textbf{Table S1} .
Crystallographic data for RLF.\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\begin{minipage}[b]{0.47\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.47\columnwidth}\raggedright\strut
\textbf{RLF}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{Data Collection}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Unit-cell parameters (\selectlanguage{ngerman}Å, \textsuperscript{o})\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{a}=58.95, \emph{b}=26.14, \emph{c}=82.87, \selectlanguage{greek}\emph{β}\selectlanguage{english}=95.5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Space group\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
\emph{P}2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Resolution (\selectlanguage{ngerman}Å)\textsuperscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
45.79-1.55 (1.58-1.55)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Wavelength (\selectlanguage{ngerman}Å)\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
1.0000\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Temperature (K)\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
100\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Observed reflections\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
150,129\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Unique reflections\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
36,463\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
\textless{}I/\selectlanguage{greek}σ\selectlanguage{english}(I)\textgreater{}\textsuperscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
11.0 (2.0)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Completeness (\%)\textsuperscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
97.6 (98.1)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Multiplicity\textsuperscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
4.1 (4.0)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
\emph{R}\textsubscript{merge} (\%)\textsuperscript{1, 2}\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
7.1 (66.6)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
\emph{R}\textsubscript{meas} (\%)\textsuperscript{1, 4}\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
8.1 (76.6)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
\emph{R}\textsubscript{pim} (\%)\textsuperscript{1, 4}\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
3.9 (37.1)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
CC\textsubscript{1/2} \textsuperscript{1, 5}\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
0.998 (0.763)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{Refinement}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Resolution (\selectlanguage{ngerman}Å) \textsuperscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
41.25-1.55\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Reflections (working/test)\textsuperscript{1}\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
34,634/1,819\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
\emph{R}\textsubscript{factor} / \emph{R}\textsubscript{free}
(\%)\textsuperscript{1,3}\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
17.5/20.0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
No. of atoms (Protein/Heme/Water)\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
1,686 / 86 / 164\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{Model Quality}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
R.m.s deviations\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Bond lengths (\selectlanguage{ngerman}Å)\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
0.008\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Bond angles (\textsuperscript{o})\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
0.961\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Mean \emph{B}-factor (\selectlanguage{ngerman}Å\textsuperscript{2})\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
All Atoms\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
23.5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Protein\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
22.9\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Heme\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
20.2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Water\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
31.3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Coordinate error\\
(maximum likelihood) (\selectlanguage{ngerman}Å)\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
0.12\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Ramachandran Plot\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Most favored (\%)\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
97.6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Additionally allowed (\%)\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
2.4\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Values in parenthesis are for the highest resolution shell.

\emph{R} \textsubscript{merge} = \selectlanguage{greek}Σ\selectlanguage{english}\emph{\textsubscript{hkl}}
\selectlanguage{greek}Σ\selectlanguage{english}\emph{\textsubscript{i}}\textbar{}\emph{I\textsubscript{i}} (\emph{hkl}
) - \textless{}\emph{I} (\emph{hkl} )\textgreater{}\textbar{} /
\selectlanguage{greek}Σ\selectlanguage{english}\emph{\textsubscript{hkl}} \selectlanguage{greek}Σ\selectlanguage{english}\emph{\textsubscript{i}I\textsubscript{i}}
(\emph{hkl} ), where\emph{I\textsubscript{i}} (\emph{hkl} ) is the
intensity

measured for the \emph{i}th reflection and
\textless{}\emph{I}(\emph{hkl})\textgreater{} is the average intensity
of all reflections with indices hkl.

\emph{R} \textsubscript{factor} = \selectlanguage{greek}Σ\selectlanguage{english}\textsubscript{\emph{hkl}}
\textbar{}\textbar{}\emph{F} \textsubscript{obs} (\emph{hkl} )
\textbar{} - \textbar{}\emph{F} \textsubscript{calc} (\emph{hkl} )
\textbar{}\textbar{} / \selectlanguage{greek}Σ\selectlanguage{english}\textsubscript{\emph{hkl}} \textbar{}\emph{F}
\textsubscript{obs} (\emph{hkl} )\textbar{}; Rfree is calculated in an

identical manner using 5\% of randomly selected reflections that were
not included in the refinement.

\emph{R} \textsubscript{meas} = redundancy-independent
(multiplicity-weighted)\emph{R}
\textsubscript{merge}.\emph{\textsuperscript{15, 67}} \emph{R}
\textsubscript{pim} = precision-indicating
(multiplicity-weighted)\emph{R}
\textsubscript{merge}.\emph{\textsuperscript{68, 69}}

CC\textsubscript{1/2} is the correlation coefficient of the mean
intensities between two random half-sets of
data.\emph{\textsuperscript{70, 71}}



\textbf{FIGURE LEGENDS}

\textbf{Figure 1.} (A ) Schematic diagram of Ncb5or constructs. Except
Ncb5or-\selectlanguage{greek}Δ\selectlanguage{english}34, all are used in the current study. (B ) Amino acid sequences
of the N-terminal region in human Ncb5or and its point-mutation variant.

\textbf{Figure 2.} Far-UV (A ) and near-UV/visible (B ) CD spectra of
Ncb5or-N/b5 and Ncb5or-b5. Far UV (C ) and near-UV/visible (D ) spectra
of Ncb5or-N as a stand-alone peptide (alone) or when fused to Ncb5or-b5
(difference, N/b5 -- b5). Difference far UV (E, G ) and near-UV/visible
(F, H ) CD spectra of Ncb5or-N/b5 variants. Difference spectra were
presented by smoothing raw data, which are shown in \textbf{Figure S3} ,
with the use of negative exponential program in SigmaPlot 10. Molar
ellipticity is used in all CD spectra. Values of signature bands are
summarized in \textbf{Table 1} .

\textbf{Figure 3.} (A ) Comparison of the domain arrangements in Ncb5or
and RLF. (B ) Amino acid sequence alignment of human Ncb5or (residues 1
-- 137), rice RLF (Oryza sativa, residues 72 -- 218), and baker's yeast
IRC21 (\emph{Saccharomyces cerevisiae,} residues 17-201). Identical and
similar residues are coded in yellow and green, respectively.

\textbf{Figure 4. (\emph{A})} Structure of RLF (subunit A) with the
secondary structure elements annotated. The heme molecule is rendered as
gold cylinders. \textbf{(\emph{B})} RLF sequence with the secondary
structure elements annotated. The red arrows indicate the His residues
that coordinate the heme.

\textbf{Figure 5.} Superposition of rice RLF (magenta) with the Ncb5or
b5-domain (PDB 3LF5, green). RMSD deviation between Ca for b5 core
residues (see Figure 4) is 1.01 \selectlanguage{ngerman}Å.

\textbf{Figure 6. (\emph{A})} Comparison of conserved, surface exposed
tryptophan residue in Ncb5or (green) and RLF (magenta).
Trp\textsuperscript{199} in the RLF structure forms a hydrogen bond with
a heme propionate group, while Trp\textsuperscript{114} in Ncb5or
extends into solvent. This flexibility suggests a possible role in
modulating electron flow. \textbf{(\emph{B})} Comparison of the heme
orientation and the coordinating His residues.
His\textsuperscript{89}/His\textsuperscript{112} are colored green for
3LF5 and His\textsuperscript{174}/His\textsuperscript{197} are colored
magenta for RLF.

\textbf{Figure 7.} View of the RLF structure highlighting helix \selectlanguage{greek}α\selectlanguage{english}1 and
locations/interactions of residues. Corresponding Ncb5or residues are
shown in parentheses. Black dotted lines indicate hydrogen bonds.

\textbf{Figure 8.} Residue Trp\textsuperscript{120} in RLF,
corresponding to Trp\textsuperscript{37} in Ncb5or, forms a network of
hydrophobic packing interactions with other conserved residues (shown
with carbon atoms rendered in yellow). The hydroxyl group of
Tyr\textsuperscript{170} (Tyr\textsuperscript{85} in Ncb5or) makes van
der Waals contact with the heme, and the thiol group of
Cys\textsuperscript{209} (Cys\textsuperscript{124} in Ncb5or) makes van
der Waals contact with Trp\textsuperscript{120}.

\textbf{Figure S1.} Sequence alignment of N-terminal region and
cytochrome b5 domain of known Ncb5or proteins of a diverse background.
Asterisk and ``\_'' indicate conserved and chemically similar residues ,
among mammals, respectively. Non-conserved residues at key positions are
indicated in red. Species (GenBank accession number) are as follows:
(mammals in yellow) human (NM\_016230), monkey (XP\_518614.3), monkey
Old World (XP\_011920356.1), guinea pig (XP\_005006001), mouse
(NM\_024195), rat (NM\_133427), cow (NM\_001038159), horse
(XM\_001499913), sheep (XP\_027818354), pig (XP\_013848110), dog
(XM\_532219), minke whale (XM\_007165020.1), ferret (XP\_004773650),
bat/Molossus (XP\_036104100), bat/Pterpus (XP\_011363184), brown bat
(XM\_008145048), opossum (XM1375853), platpus (XP\_028903113); (birds,
reptiles and amphibians in green) Zebra Finch (XP\_030123394), golden
eagle (XP\_029856007), peregrine falcon (XP\_013152060), chicken
(XM\_001233870), Dalmatian pelican (KFQ624870), crested ibis (KFR09350),
seriema (KFP68580), swan goose (XM\_013180133), turkey (XP\_010705963),
turtle/Terrapene (XP\_026510156) turtle/Pelodiscus (XP\_006133410),
garter snake (XP\_032072321), Xenopus (XP\_031757575), Coelacanth
(XP\_014343350); (fishes in blue) Cyprinidae (XM\_016550377), Zebrafish
(NP\_001018496), Pimephales (XP\_039507272), Salmon (XP\_020361560),
bony fish (XM\_008279520), yellow croaker (XM\_019265374), Fugu
(TNN02071), Pufferfish/Japan (XM\_003965751), tire track eel
(XR\_003294861), swamp eel (XM\_020621659), Gasterosteus
(XP\_040020873), rice fish (AM140533), rice fish India (XP\_024117733),
Lamprey (XP\_032829495), Skate (XP\_032877801), seahorse
(XM\_019880106), (invertebrates in grey) Lancelet (XM\_002603916),
Trichoplax (XM\_002112883), tunicate (XR\_053035), Hydra
(XM\_002165807), sea anemone (XP\_048577331), coral (EZ038676,
Acropora), coral/Exaiptasia (KXJ05517), sea urchin/Lytechinus
(XP\_041463305), sea urchin (XP1198870), Teladorsagia (PIO73582),
hookworm/Necator (RCN51358), C.elegans (NM\_001026613), pig worm
(KFD50571c, Trichuris suis), Trichuris (KHJ45328); (insects in purple)
Drosophila (NM\_137575), Mosquito/Anopheles (XP\_041781397),
Mosquito/Aedes (KXJ83737), Honeybee (XM\_394412), Louse (XM\_002428283),
Jewel wasp (XM\_001601866), Beetle (XM\_963135), Pea aphid
(XM\_001948299), Deer tick (XM\_002401084),

\textbf{Figure S2.} Prediction of disordered domains in Ncb5or by PONDR
VL-XT (A ). Prediction of secondary structure in the 50 N-terminal
residues of Ncb5or by PSIPRED and PROF (B ) and by AGADIR (C ). AA
(amino acid): Target sequence, Pred: Predicted secondary structure
(H=helix, E=strand, C=coil), Conf: Confidence (0=low, 9=high).

\textbf{Figure S3.} Sequence alignment of the N-terminal region and b5
core (partial) of human Ncb5or against RLF proteins in plants. Asterisk
(*) indicates conserved residues, whereas ``\_'' represents chemically
similar residues.

\textbf{Figure S4} . Sequence alignment of the N-terminal region of
human Ncb5or against IRC21 proteins in fungi. Asterisk (*) indicates
conserved residues, whereas ``\_'' represents chemically similar
residues.

\textbf{Figure S5} . Sequence alignment of the N-terminal region and
cytochrome b5 domain of human Ncb5or against cytochrome b5 proteins in
protists (\emph{Dictyostelium} and \emph{Trypanosoma} ) and parasites
(all others). Asterisk (*) indicates conserved residues, whereas ``;''
represents chemically similar residues. The conserved ``HPGG'' heme
binding site is highlighted, and the N-terminal motif in human and
protists is indicated by ``------``.

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