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

NADH cytochrome b₅ oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b₅ (b₅ ) and cytochrome b₅ reductase (b₅ R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain 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 b₅ R domains (CS/b₅ R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L³⁴ MDWIRL⁴⁰ motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching 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 (S¹¹⁶ QMDWLKLTRT¹²⁶ ) 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 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 multi-domain 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.

Keywords: circular dichroism; crystallography; cytochrome b5; helix motif; intrinsically disordered region.