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The N-terminal intrinsically disordered region of Ncb5or docks with the cytochrome b5 core to form a helical motif that is of ancient origin
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  • Hao Zhu,
  • David Benson,
  • Bin Deng,
  • Maithri M. Kashipathy,
  • Scott Lovell,
  • Kevin P. Battaile,
  • Anne Cooper,
  • Philip Gao,
  • Aron W. Fenton
Hao Zhu
The University of Kansas Medical Center

Corresponding Author:[email protected]

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David Benson
The University of Kansas
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Bin Deng
The University of Kansas Medical Center
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Maithri M. Kashipathy
The University of Kansas
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Scott Lovell
The University of Kansas
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Kevin P. Battaile
New York Structural Biology Center
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Anne Cooper
The University of Kansas
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Philip Gao
The University of Kansas
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Aron W. Fenton
The University of Kansas Medical Center
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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 34MDWIRL 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 α-helix (S 116QMDWLKLTRT 126) 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 (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.
22 Jul 2023Submitted to PROTEINS: Structure, Function, and Bioinformatics
22 Jul 2023Submission Checks Completed
22 Jul 2023Assigned to Editor
22 Jul 2023Review(s) Completed, Editorial Evaluation Pending
31 Jul 2023Reviewer(s) Assigned
19 Sep 2023Editorial Decision: Revise Minor
27 Oct 20231st Revision Received
27 Oct 2023Assigned to Editor
27 Oct 2023Submission Checks Completed
27 Oct 2023Review(s) Completed, Editorial Evaluation Pending
29 Oct 2023Reviewer(s) Assigned
09 Nov 2023Editorial Decision: Revise Minor
11 Nov 20232nd Revision Received
11 Nov 2023Submission Checks Completed
11 Nov 2023Assigned to Editor
11 Nov 2023Review(s) Completed, Editorial Evaluation Pending
17 Nov 2023Editorial Decision: Accept