Conformational Change of the Hairpin-like-structured Robo2 Ectodomain Allows NELL1/2 Binding
Conformational Change of the Hairpin-like-structured Robo2 Ectodomain Allows NELL1/2 Binding
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发夹状结构 Robo2 胞外域的构象变化允许 NELL1/2 结合
DOI：
10.1016/j.jmb.2022.167777
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发表时间：
2022
影响因子：
5.6
通讯作者：
Niimi Tomoaki
中科院分区：
文献类型：
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作者：
Miyaguchi Masaki;Nakanishi Yoichi;Maturana Andr?s D.;Mizutani Kimihiko;Niimi Tomoaki
自从神经表皮生长因子样 (NELL) 2 被确定为环形 (Robo) 3 受体的新型配体以来，对 NELL-Robo 信号传导的研究变得越来越重要。我们之前报道过 Robo2 在酸性条件下可以与 NELL1/2 结合，但在中性 pH 条件下则不能结合。在中性条件下被封闭的 NELL1/2 结合位点被认为是由于 Robo2 胞外域在暴露于酸性 pH 值时发生构象变化而暴露出来的。然而，其潜在的结构机制尚不清楚。在这里，我们研究了形成 Robo2 胞外域发夹样结构的免疫球蛋白样结构域和 III 型纤连蛋白结构域之间的相互作用，并证明酸性 pH 会减弱它们之间的相互作用。 Robo2 的选择性剪接异构体会影响发夹样结构的构象，被发现具有不同的 NELL1/2 结合亲和力。我们开发了基于 Förster 共振能量转移的指示器，通过在 Robo2 胞外域末端单独插入供体和受体荧光蛋白来监测其构象变化。这些实验表明，Robo2 胞外域的末端在酸性条件下彼此靠近。通过将这些发现与尺寸排阻色谱分析的结果相结合，我们认为，在酸性条件下，Robo2 胞外域具有紧凑的构象和松散的发夹状结构。这些结果可能有助于阐明 Robo2 和 NELL1/2 在酸性条件下相互作用产生的信号传导机制。
Since neural epidermal growth factor-like-like (NELL) 2 was identified as a novel ligand for the roundabout (Robo) 3 receptor, research on NELL–Robo signaling has become increasingly important. We have previously reported that Robo2 can bind to NELL1/2 in acidic conditions but not at neutral pH. The NELL1/2-binding site that is occluded in neutral conditions is thought to be exposed by a conformational change of the Robo2 ectodomain upon exposure to acidic pH; however, the underlying structural mechanisms are not well understood. Here, we investigated the interaction between the immunoglobulin-like domains and fibronectin type III domains that form hairpin-like structure of the Robo2 ectodomain, and demonstrated that acidic pH attenuates the interaction between them. Alternative splicing isoforms of Robo2, which affect the conformation of the hairpin-like structure, were found to have distinct NELL1/2-binding affinities. We developed Förster resonance energy transfer-based indicators for monitoring conformational change of the Robo2 ectodomain by individually inserting donor and acceptor fluorescent proteins at its ends. These experiments revealed that the ends of the Robo2 ectodomain are close to each other in acidic conditions. By combining these findings with the results of size exclusion chromatography analysis, we suggest that, in acidic conditions, the Robo2 ectodomain has a compact conformation with a loose hairpin-like structure. These results may help elucidate the signaling mechanisms resulting from the interaction between Robo2 and NELL1/2 in acidic conditions.