High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface
High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface
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DOI：
10.1074/jbc.m115.648410
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发表时间：
2015-04-24
影响因子：
4.8
通讯作者：
Sharp, Joshua S.
中科院分区：
文献类型：
--
作者：
Li, Zixuan;Moniz, Heather;Sharp, Joshua S.
Robo家族的跨膜受体和分泌蛋白Slit的相互作用在中枢神经系统的发育和轴突中线穿越的调节中提供重要信号。硫酸乙酰肝素是一种以多种复杂方式修饰的硫酸化线性多糖，在Slit-Robo信号传导中充当重要的辅助受体。先前的研究表明，密切相关的肝素八糖直接结合果蝇Robo，表面等离子体共振分析显示，Robo 1更紧密地结合全长未分级肝素。对于第一次，我们利用电子转移解离为基础的高空间分辨率的羟基自由基蛋白足迹识别两个单独的结合位点的肝素相互作用与Robo 1：一个结合位点在先前确定的网站肝素DP 8和第二个结合位点在N末端的Robo 1是无序的X-射线晶体结构。通过表面等离子体共振和肝素亲和色谱法测定，所鉴定的N-末端结合位点的突变表现出结合亲和力的降低。足迹法还表明，肝素结合诱导的Ig 2结构域的构象和/或动力学的微小变化，但没有检测到主要的构象变化。这些结果表明Robo-Slit复合物中存在第二个低亲和力结合位点，并表明Robo 1的Ig 2结构域在肝素介导的信号转导中的作用。这项研究也标志着第一次使用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，这表明蛋白质-碳水化合物复合物的表征具有很大的实用性。
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.