CONCLUSION
This study represents the first 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
interactions54-57 , of which
the specificity is generally mediated by motif
structures.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.47Intrinsically disordered regions appear to be enriched in
disease-related proteins, such as the N-terminal transactivation domain
of tumor suppressor
p53,60alpha-synuclein in Parkinson’s
disease61 , tau
protein in Alzheimer’s
disease62 , and
in a number of proteins associated with cardiovascular
disease.63Mutation 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.64Functional 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
ago48,
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.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 α1 from α2 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.66Functional roles could include modulating heme reduction by the b5R
domain or reduction by heme of the still unknown substrate.