Structural Aspects of Heparan Sulfate Binding to Robo1-Ig1-2
Structural Aspects of Heparan Sulfate Binding to Robo1-Ig1-2
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DOI：
10.1021/acschembio.6b00692
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发表时间：
2016-11-01
影响因子：
4
通讯作者：
Prestegard, James H.
中科院分区：
文献类型：
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作者：
Gao, Qi;Chen, Cheng-Yu;Prestegard, James H.
Roundabout 1，或Robo 1，是一种在轴突导向中重要的细胞表面信号分子。其与硫酸乙酰肝素（HS）和Slit蛋白家族成员的相互作用对其活性至关重要，因此非常需要通过结构方法（如NMR）表征这些相互作用。然而，Robo 1是糖基化蛋白的事实阻止了使用常用的细菌宿主来表达具有NMR研究所需的均匀N-15、C-13和H-2标记的适当糖基化形式。在这里，我们应用了一种替代方法，基于用单一氨基酸类型和高结构含量NMR数据标记，来表征糖基化Robo 1（Robo 1-Ig 1 -2）与合成HS四聚体（IdoA-GlcNS 6S-wIdoA 2S-GlcNS 6S-（CH 2）（5）NH 2）相互作用的双结构域构建体。显着的化学位移扰动的交叉口从K81滴定与四聚体的结合位点的位置提供了初步的证据，并允许确定一个255 μ M的解离常数。HS四聚体的结合表位、结合构象和结合位点位置已通过饱和转移差异（STD）、转移核Overhauser效应（trNOE）和顺磁扰动实验进一步表征。复杂的模型已产生使用来自各种NMR实验的约束。该模型的后处理能量分析提供了每个聚糖残基在结合事件中所起作用的基本原理，并且在先前Robo-Slit结构的背景下检查结合位点提供了HS调节Robo-Slit相互作用的基本原理。
Roundabout 1, or Robo1, is a cell surface signaling molecule important in axon guidance. Its interaction with heparan sulfate (HS) and members of the Slit protein family is essential to its activity, making characterization of these interactions by structural methods, such as NMR, highly desirable. However, the fact that Robo1 is a glycosylated protein prevents employment of commonly used bacterial hosts for expression of properly glycosylated forms with the uniform N-15, C-13, and H-2 labeling needed for NMR studies. Here, we apply an alternative methodology, based on labeling with a single amino acid type and high structural content NMR data, to characterize a two-domain construct of glycosylated Robo1 (Robo1-Ig1-2) interacting with a synthetic HS tetramer (IdoA-GlcNS6S-wIdoA2S-GlcNS6S-(CH2)(5)NH2). Significant chemical shift perturbations of the crosspeak from K81 on titration with the tetramer provide initial evidence for the location of a binding site and allow determination of a 255 mu M disassociation constant. The binding epitopes, bound conformation, and binding site placement of the HS tetramer have been further characterized by saturation transfer difference (STD), transferred nuclear Overhauser effect (trNOE), and paramagnetic perturbation experiments. A model of the complex has been generated using constraints derived from the various NMR experiments. Postprocessing energetic analysis of this model provides a rationale for the role each glycan residue plays in the binding event, and examination of the binding site in the context of a previous Robo-Slit structure provides a rationale for modulation of Robo-Slit interactions by HS.