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.