8TGB

Crystal structure of root lateral formation protein (RLF) b5-domain from Oryza sativa

Summary for 8TGB

• Entry DOI: 10.2210/pdb8tgb/pdb
• Descriptor: root lateral formation protein (RLF), PROTOPORPHYRIN IX CONTAINING FE, SULFATE ION, ... (4 entities in total)
• Functional Keywords: root lateral formation protein, ncb5or-b5 domain, cytochrome b5 heme-binding, oryza sativa, hydrolase, oxidoreductase
• Biological source: Oryza sativa Japonica Group (Japanese rice)
• Total number of polymer chains: 2
• Total formula weight: 28735.09

Authors

Lovell, S.,Kashipathy, M.M.,Battaile, K.P.,Benson, D.R. (deposition date: 2023-07-12, release date: 2023-12-13, Last modification date: 2024-03-20)

Primary citation

Benson, D.R.,Deng, B.,Kashipathy, M.M.,Lovell, S.,Battaile, K.P.,Cooper, A.,Gao, P.,Fenton, A.W.,Zhu, H.
The N-terminal intrinsically disordered region of Ncb5or docks with the cytochrome b 5 core to form a helical motif that is of ancient origin.
Proteins, 92:554-566, 2024

PubMed Abstract: NADH cytochrome b oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b (b ) and cytochrome b reductase (b 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 b domain and a construct containing the CS and b R domains (CS/b 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 L MDWIRL 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 b domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b 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 Trp (corresponding to invariant Trp in Ncb5or) to be part of an 11-residue α-helix (S QMDWLKLTRT ) packing against two of the four helices in the b domain that surround heme (α2 and α5). The Trp side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr and Tyr (α2), Cys (α5), and Leu in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp in nucleating the formation of the N-terminal helix, whose location in the N/b 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/b module as reflected by its existence in a class of cytochrome b proteins from three kingdoms among eukaryotes.

PubMed: 38041394
DOI: 10.1002/prot.26647

Experimental method

X-RAY DIFFRACTION (1.55 Å)