Department of Plant and Microbial Biology (IPMB), Zurich-Basel Plant Science Center, University of Zurich, Zurich 8008, Switzerland.
The Plant Signaling Mechanisms Laboratory, Department of Plant Molecular Biology, University of Lausanne, Lausanne 1015, Switzerland.
Proc Natl Acad Sci U S A. 2024 Aug 13;121(33):e2400862121. doi: 10.1073/pnas.2400862121. Epub 2024 Aug 6.
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 . 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.
分泌信号肽是生长、发育和应激反应的核心调节剂，但这些肽及其受体的进化特定步骤尚不清楚。此外，由于全球少数实验室拥有有限的蛋白质结构测定能力，因此仅对少数几个例子的肽-受体结合的分子机制有所了解。植物已经进化出多种分泌信号肽和相应的跨膜受体。最近鉴定出应激响应的富含丝氨酸的内源性肽（SCOOPs）。生物活性 SCOOPs 被枯草杆菌蛋白酶亚基切割，并被模式植物中的富含亮氨酸重复受体激酶 MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2)感知。SCOOPs 和 MIK2 如何（共同）进化，以及 SCOOPs 如何与 MIK2 结合尚不清楚。我们通过对 350 种植物基因组进行计算机分析，随后进行功能测试，揭示了 MIK2 作为 Brassicales 植物目中 SCOOP 受体的保守性。然后，我们利用基于人工智能的结构建模和比较基因组学，鉴定出 Brassicales MIK2 同源物中两个保守的假定 SCOOP-MIK2 结合口袋，这些口袋被预测与 otherwise sequence-divergent SCOOPs 的“SxS”基序相互作用。两个预测的结合口袋的突变均削弱了 SCOOP 与 MIK2 的结合，SCOOP 诱导 MIK2 与其核心受体 BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 之间的复合物形成，以及 SCOOP 诱导的活性氧产生，从而证实了我们的计算机预测。总的来说，除了揭示难以捉摸的 SCOOP-MIK2 结合机制外，我们的分析流程结合了系统发生基因组学、基于人工智能的结构预测以及实验生物化学和生理验证，为阐明肽配体-受体感知机制提供了蓝图。
