PDF(Pubmed)
Abstract：
Secreted signaling peptides are central regulators of growth, development, and stress responses, but specific steps in the evolution of these peptides and their receptors are not well understood. Also, the molecular mechanisms of peptide-receptor binding are only known for a few examples, primarily owing to the limited availability of protein structural determination capabilities to few laboratories worldwide. Plants have evolved a multitude of secreted signaling peptides and corresponding transmembrane receptors. Stress-responsive SERINE RICH ENDOGENOUS PEPTIDES (SCOOPs) were recently identified. Bioactive SCOOPs are proteolytically processed by subtilases and are perceived by the leucine-rich repeat receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) in the model plant Arabidopsis thaliana. How SCOOPs and MIK2 have (co)evolved, and how SCOOPs bind to MIK2 are unknown. Using in silico analysis of 350 plant genomes and subsequent functional testing, we revealed the conservation of MIK2 as SCOOP receptor within the plant order Brassicales. We then leveraged AI-based structural modeling and comparative genomics to identify two conserved putative SCOOP-MIK2 binding pockets across Brassicales MIK2 homologues predicted to interact with the \"SxS\" motif of otherwise sequence-divergent SCOOPs. Mutagenesis of both predicted binding pockets compromised SCOOP binding to MIK2, SCOOP-induced complex formation between MIK2 and its coreceptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1, and SCOOP-induced reactive oxygen species production, thus, confirming our in silico predictions. Collectively, in addition to revealing the elusive SCOOP-MIK2 binding mechanism, our analytic pipeline combining phylogenomics, AI-based structural predictions, and experimental biochemical and physiological validation provides a blueprint for the elucidation of peptide ligand-receptor perception mechanisms.
摘要：
分泌的信号肽是生长的中心调节因子，发展,和应激反应，但是这些肽及其受体进化的具体步骤还没有得到很好的理解。此外，肽-受体结合的分子机制只有几个例子是已知的，主要是由于全球很少的实验室对蛋白质结构测定能力的可用性有限。植物已经进化出大量分泌的信号肽和相应的跨膜受体。应激反应性丝氨酸富内源性肽(SCOOPs)最近被鉴定。生物活性SCOOP被枯草杆菌酶蛋白水解处理,并被模型植物拟南芥中富含亮氨酸的重复受体激酶男性发现因子1-相互作用受体样激酶2(MIK2)感知。SCOOP和MIK2是如何(共同)进化的，以及SCOOP如何与MIK2结合是未知的。使用350个植物基因组的计算机模拟分析和随后的功能测试，我们揭示了MIK2作为SCOOP受体的保守性。然后，我们利用基于AI的结构建模和比较基因组学来鉴定两个保守的假定SCOOP-MIK2结合口袋，这些同源物预测与序列不同的SCOOP的“SxS”基序相互作用。两个预测的结合口袋的诱变损害了SCOOP与MIK2的结合，SCOOP诱导的MIK2与其共受体的胆碱酯酶不敏感1相关激酶1之间的复合物形成，以及SCOOP诱导的活性氧产生，因此，证实了我们的预测.总的来说，除了揭示难以捉摸的SCOOP-MIK2结合机制外，我们的分析管道结合了系统基因组学，基于人工智能的结构预测，实验生化和生理验证为阐明肽配体-受体感知机制提供了蓝图。
