School of Molecular Sciences, The University of Western Australia, Crawley, Perth 6009, Australia.
The ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Perth 6009, Australia.
Plant Cell. 2021 Aug 31;33(8):2794-2811. doi: 10.1093/plcell/koab130.
Over 30 years ago, an intriguing posttranslational modification was found responsible for creating concanavalin A (conA), a carbohydrate-binding protein from jack bean (Canavalia ensiformis) seeds and a common carbohydrate chromatography reagent. ConA biosynthesis involves what was then an unprecedented rearrangement in amino-acid sequence, whereby the N-terminal half of the gene-encoded conA precursor (pro-conA) is swapped to become the C-terminal half of conA. Asparaginyl endopeptidase (AEP) was shown to be involved, but its mechanism was not fully elucidated. To understand the structural basis and consequences of circular permutation, we generated recombinant jack bean pro-conA plus jack bean AEP (CeAEP1) and solved crystal structures for each to 2.1 and 2.7 Å, respectively. By reconstituting conA biosynthesis in vitro, we prove CeAEP1 alone can perform both cleavage and cleavage-coupled transpeptidation to form conA. CeAEP1 structural analysis reveals how it is capable of carrying out both reactions. Biophysical assays illustrated that pro-conA is less stable than conA. This observation was explained by fewer intermolecular interactions between subunits in the pro-conA crystal structure and consistent with a difference in the prevalence for tetramerization in solution. These findings elucidate the consequences of circular permutation in the only posttranslation example known to occur in nature.
30 多年前，人们发现了一种有趣的翻译后修饰，它负责产生伴刀豆球蛋白 A（conA），这是一种来自刀豆（Canavalia ensiformis）种子的碳水化合物结合蛋白，也是一种常见的碳水化合物色谱试剂。ConA 的生物合成涉及到当时前所未有的氨基酸序列重排，即基因编码的 conA 前体（pro-conA）的 N 端半部分被交换成为 conA 的 C 端半部分。天冬酰胺内肽酶（AEP）被证明参与其中，但它的机制尚未完全阐明。为了理解环状排列的结构基础和后果，我们生成了重组刀豆 pro-conA 加上刀豆 AEP（CeAEP1），并分别将其晶体结构解析至 2.1 和 2.7 Å。通过在体外重新构建 conA 生物合成，我们证明 CeAEP1 可以单独进行切割和切割偶联转肽反应以形成 conA。CeAEP1 的结构分析揭示了它如何能够进行这两种反应。生物物理测定表明 pro-conA 不如 conA 稳定。这种观察结果可以通过在 pro-conA 晶体结构中亚基之间的分子间相互作用较少来解释，并且与溶液中四聚体形成的普遍性差异一致。这些发现阐明了环状排列在自然界中已知的唯一的翻译后发生的后果。
