Structural signatures of the class III lasso peptide BI-32169 and the branched-cyclic topoisomers using trapped ion mobility spectrometry-mass spectrometry and tandem mass spectrometry
Structural signatures of the class III lasso peptide BI-32169 and the branched-cyclic topoisomers using trapped ion mobility spectrometry-mass spectrometry and tandem mass spectrometry
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DOI：
10.1007/s00216-019-01613-8
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发表时间：
2019-09-01
影响因子：
4.3
通讯作者：
Fernandez-Lima, Francisco
中科院分区：
文献类型：
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作者：
Fouque, Kevin Jeanne Dit;Bisram, Vikash;Fernandez-Lima, Francisco
Lasso肽是一类具有生物活性的核糖体合成和后修饰的肽（RIPPs），其特征在于机械互锁拓扑结构，其中肽的C-末端尾被穿入并捕获在N-末端大环内酰胺环内。BI-32169是一种III类套索肽，含有一个二硫键，可进一步稳定套索结构。与其支链环状类似物相比，BI-32169具有更高的稳定性，已知对人胰高血糖素受体具有强效抑制活性。在目前的工作中，串联质谱，使用碰撞诱导解离（CID）和电子捕获解离（ECD），和捕获离子迁移谱-质谱（TIMS-MS）实验进行了证据的两个拓扑结构的特定结构签名。CID实验显示两种拓扑异构体的片段化模式相似，其中C-末端尾部的一部分通过二硫键与大环内酰胺环保持共价连接，这不能清楚地构成套索拓扑结构的特征。BI-32169的ECD实验显示，与其支链环状拓扑异构体相比，环区域中的氢迁移事件增加，证明了套索拓扑结构的特定结构特征。TIMS的高迁移率分辨能力导致质子化物种的多种构象的识别，但不允许在混合物中的两种拓扑结构的明确区分。当与铯金属离子络合时，构象的数量减少导致两种结构的明确识别。还原和烷基化BI-32169二硫键的实验表明，套索结构得以保留且具有热稳定性，相关构象变化为二硫键在人胰高血糖素受体抑制活性中的作用提供了新的见解。
Lasso peptides are a class of bioactive ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked topology, where the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. BI-32169 is a class III lasso peptide containing one disulfide bond that further stabilizes the lasso structure. In contrast to its branched-cyclic analog, BI-32169 has higher stability and is known to exert a potent inhibitory activity against the human glucagon receptor. In the present work, tandem mass spectrometry, using collision-induced dissociation (CID) and electron capture dissociation (ECD), and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) experiments were carried out to evidence specific structural signatures of the two topologies. CID experiments showed similar fragmentation patterns for the two topoisomers, where a part of the C-terminal tail remains covalently linked to the macrolactam ring by the disulfide bond, which cannot clearly constitute a signature of the lasso topology. ECD experiments of BI-32169 showed an increase of hydrogen migration events in the loop region when compared with those of its branched-cyclic topoisomer evidencing specific structural signatures for the lasso topology. The high mobility resolving power of TIMS resulted in the identification of multiple conformations for the protonated species but did not allow the clear differentiation of the two topologies in mixture. When in complex with cesium metal ions, a reduced number of conformations led to a clear identification of the two structures. Experiments reducing and alkylating the disulfide bond of BI-32169 showed that the lasso structure is preserved and heat stable and the associated conformational changes provide new insights about the role of the disulfide bond in the inhibitory activity against the human glucagon receptor.