We are entering an exciting time in structural biology where artificial intelligence can be used to predict protein structures with greater accuracy than ever before. Extending this level of accuracy to the predictions of disulfide-rich peptide structures is likely to be more challenging, at least in the short term, given the tight packing of cysteine residues and the numerous ways that the disulfide bonds can potentially be linked. It has been previously shown in many cases that several disulfide bond connectivities can be accommodated by a single set of NMR-derived structural data without significant violations. Disulfide-rich peptides are prevalent throughout nature, and arguably the most well-known are those present in venoms from organisms such as cone snails. Here, we have determined the first three-dimensional structure and disulfide connectivity of a U-superfamily cone snail venom peptide, TxVIIB. TxVIIB has a VI/VII cysteine framework that is generally associated with an inhibitor cystine knot (ICK) fold; however, AlphaFold predicted that the peptide adopts a mini-granulin fold with a granulin disulfide connectivity. Our experimental studies using NMR spectroscopy and orthogonal protection of cysteine residues indicate that TxVIIB indeed adopts a mini-granulin fold but with the ICK disulfide connectivity. Our findings provide structural insight into the underlying features that govern formation of the mini-granulin fold rather than the ICK fold and will provide fundamental information for prediction algorithms, as the subtle complexity of disulfide isomers may be not adequately addressed by the current prediction algorithms.

中文翻译：

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含有小颗粒蛋白折叠的 U 超家族芋螺毒素的结构分析：深入了解区分 ICK 和颗粒蛋白折叠的关键特征

我们正在进入结构生物学的一个激动人心的时代，人工智能可以比以往任何时候都更准确地预测蛋白质结构。考虑到半胱氨酸残基的紧密堆积以及二硫键可能连接的多种方式，将这种准确性水平扩展到富含二硫键的肽结构的预测可能更具挑战性，至少在短期内是这样。先前在许多情况下已经表明，一组 NMR 衍生的结构数据可以容纳多个二硫键连接，而不会出现重大违规。富含二硫化物的肽在自然界中普遍存在，可以说最著名的是存在于锥蜗牛等生物体毒液中的肽。在这里，我们确定了 U 超家族锥蜗牛毒液肽 TxVIIB 的第一个三维结构和二硫键连接。 TxVIIB 具有 VI/VII 半胱氨酸框架，通常与抑制剂胱氨酸结 (ICK) 折叠相关；然而，AlphaFold 预测该肽采用具有颗粒蛋白二硫键连接的小颗粒蛋白折叠。我们使用核磁共振波谱和半胱氨酸残基正交保护的实验研究表明，TxVIIB 确实采用了小颗粒蛋白折叠，但具有 ICK 二硫键连接。我们的研究结果提供了对控制小颗粒蛋白折叠而不是 ICK 折叠形成的基本特征的结构洞察，并将为预测算法提供基本信息，因为当前的预测算法可能无法充分解决二硫键异构体的微妙复杂性。