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首页> 美国卫生研究院文献>The Journal of Biological Chemistry >High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface
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High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface

机译：高结构分辨率羟自由基蛋白质足迹揭示了扩展的Robo1-肝素结合界面。

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Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes.
机译：Robo家族的跨膜受体与分泌蛋白Slit的相互作用为中枢神经系统的发育和轴突中线穿越的调控提供了重要信号。硫酸乙酰肝素是一种以多种方式修饰的硫酸化线性多糖，可作为Slit-Robo信号转导中必不可少的辅助受体。先前的研究表明，密切相关的肝素八糖直接与果蝇Robo结合，表面等离子体共振分析表明Robo1与全长未分级肝素结合得更紧密。首次，我们利用基于电子传递解离的高空间分辨率羟基自由基蛋白足迹识别了肝素与Robo1相互作用的两个独立结合位点：一个在先前确定的肝素dp8结合位点和另一个在N结合位点X射线晶体结构无序的Robo1末端。如通过表面等离振子共振和肝素亲和层析所测量的，所鉴定的N-末端结合位点的诱变表现出结合亲和力的降低。足迹也表明，肝素结合诱导了Ig2结构域构象和/或动力学的微小变化，但未检测到主要构象变化。这些结果表明在Robo-Slit复合物中的第二个低亲和力结合位点，也暗示了Robo1的Ig2结构域在肝素介导的信号转导中的作用。这项研究还标志着基于电子转移解离的高空间分辨率羟基自由基蛋白质足迹的首次使用，这在表征蛋白质-碳水化合物复合物方面显示出巨大的实用性。

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