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 (b₅R) 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 b₅R domains (CS/b₅R). 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 L³⁴MDWIRL⁴⁰ 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 (S¹¹⁶QMDWLKLTRT¹²⁶) 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.

中文翻译：

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Ncb5or 的 N 端本质上无序区域与细胞色素 b5 核心对接，形成古老起源的螺旋基序

NADH 细胞色素b ₅氧化还原酶 (Ncb5or) 是一种胞质铁还原酶，与糖尿病和神经系统疾病有关。Ncb5or 包含由 CHORD-Sgt1 (CS) 接头结构域分隔的细胞色素b ₅ ( b ₅ ) 和细胞色素b ₅还原酶 ( b _{5 R) 结构域。}Ncb5 或氧化还原活性取决于适当的域间相互作用来介导从 NADH 或 NADPH 通过 FAD 到血红素的电子转移。虽然全长人类 Ncb5or 已被证明具有抗结晶性，但我们已成功获得b ₅结构域的高分辨率原子结构以及包含 CS 和b ₅ R 结构域 (CS/ b ₅ R) 的构建体。Ncb5or 还包含一个由 50 个残基组成的 N 端固有无序区域，该区域在动物的其他蛋白质家族中没有同源物，但具有独特、保守的 L ³⁴ MDWIRL ⁴⁰基序，该基序也存在于水稻侧根形成 (RLF) 蛋白减少和重组增加中面包酵母中的中心 21，全部连接至b ₅结构域。在尝试结晶包含自然融合到b ₅结构域的 N 端区域的人 Ncb5or 构建体失败后，我们能够获得与人 Ncb5or 残基 25-129 相对应的重组水稻 RLF 构建体的高分辨率原子结构（ 52% 序列同一性；74% 相似性）。该结构揭示了 Trp ¹²⁰（对应于 Ncb5or 中不变的 Trp ³⁷）是 11 个残基 α 螺旋 (S ¹¹⁶ QMDWLKLTRT ¹²⁶ ) 的一部分，与血红素周围的b ₅结构域中的四个螺旋中的两个（α2 和 α5）堆积在一起）。Trp ¹²⁰侧链与四个高度保守残基的侧链形成相互作用网络，这些残基对应于Ncb5or 中的Tyr ⁸⁵和 Tyr ⁸⁸ (α2)、Cys ¹²⁴ (α5) 和 Leu ^{47 。}人 Ncb5or 片段的圆二色性测量进一步支持 Trp ³⁷在 N 末端螺旋成核中的关键作用，其在 N/ b ₅模块中的位置表明在调节这种多结构域氧化还原酶的功能中发挥作用。这项研究首次揭示了 N/ b ₅模块中螺旋基序的古老起源，这反映在它存在于来自真核生物三个王国的一类细胞色素b _{5蛋白中。}