Several proteins from plant pathogenesis-related family 10 (PR10) are highly abundant in the latex of opium poppy and have recently been shown to play diverse and important roles in the biosynthesis of benzylisoquinoline alkaloids (BIAs). The recent determination of the first crystal structures of PR10-10 showed how large conformational changes in a surface loop and adjacent β-strand are coupled to the binding of BIA compounds to the central hydrophobic binding pocket. A more detailed analysis of these conformational changes is now reported to further clarify how ligand binding is coupled to the formation and cleavage of an intermolecular disulfide bond that is only sterically allowed when the BIA binding pocket is empty. To decouple ligand binding from disulfide-bond formation, each of the two highly conserved cysteine residues (Cys59 and Cys155) in PR10-10 was replaced with serine using site-directed mutagenesis. Crystal structures of the Cys59Ser mutant were determined in the presence of papaverine and in the absence of exogenous BIA compounds. A crystal structure of the Cys155Ser mutant was also determined in the absence of exogenous BIA compounds. All three of these crystal structures reveal conformations similar to that of wild-type PR10-10 with bound BIA compounds. In the absence of exogenous BIA compounds, the Cys59Ser and Cys155Ser mutants appear to bind an unidentified ligand or mixture of ligands that was presumably introduced during expression of the proteins in Escherichia coli. The analysis of conformational changes triggered by the binding of BIA compounds suggests a molecular mechanism coupling ligand binding to the disruption of an intermolecular disulfide bond. This mechanism may be involved in the regulation of biosynthetic reactions in plants and possibly other organisms.

中文翻译：

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罂粟主要乳胶蛋白中配体触发的分子间二硫键开关的结构分析。


来自植物发病机制相关家族 10 (PR10) 的几种蛋白质在罂粟乳胶中含量很高，最近已被证明在苄基异喹啉生物碱 (BIA) 的生物合成中发挥着多种重要作用。最近对 PR10-10 的第一个晶体结构的测定表明，表面环和相邻 β 链的巨大构象变化与 BIA 化合物与中央疏水性结合袋的结合有关。现在报道了对这些构象变化的更详细分析，以进一步阐明配体结合如何与分子间二硫键的形成和裂解偶联，而分子间二硫键仅在 BIA 结合袋为空时才在空间上允许。为了将配体结合与二硫键形成解耦，PR10-10 中两个高度保守的半胱氨酸残基（Cys59 和 Cys155）均通过定点诱变被丝氨酸取代。在罂粟碱存在且不存在外源 BIA 化合物的情况下测定了 Cys59Ser 突变体的晶体结构。 Cys155Ser 突变体的晶体结构也在不存在外源 BIA 化合物的情况下测定。所有这三种晶体结构都显示出与结合 BIA 化合物的野生型 PR10-10 相似的构象。在不存在外源 BIA 化合物的情况下，Cys59Ser 和 Cys155Ser 突变体似乎与未鉴定的配体或配体混合物结合，这些配体可能是在大肠杆菌中表达蛋白质期间引入的。对 BIA 化合物结合引发的构象变化的分析表明，配体结合与分子间二硫键破坏之间存在耦合的分子机制。 这种机制可能涉及植物和其他生物体中生物合成反应的调节。