Towards understanding the glycan code: next generation structural glycobiology for accurate description of protein-glycan complexes

生命系统的非凡特性是它们的生物分子成分(蛋白质、核酸、碳水化合物或多糖等)之间动态相互作用的结果。尽管数以百万计的分子相互作用系统是复杂的，但对于健康的有机体来说，整个相互作用网络受到高度调控，调控过程中的变化是所有疾病的基础。在这个网络中，不同的分子以不同的强度(亲和力)相互作用，其中强相互作用的伙伴产生稳定的生物分子复合体，弱结合的分子产生瞬时组装。一个非常不同亲和力的蛋白质-配体相互作用的良好“协调”系统确实负责生物体内的大部分调控过程。当需要持续的生物信号时，形成强复合体，而当需要临时刺激(信号转导、可逆的细胞-细胞接触、宿主/病原体识别的瞬时相互作用等)后需要快速细胞反应时，弱相互作用被唤起。为了完整地了解生命过程，有必要研究蛋白质-配体的强相互作用和弱相互作用，这促进了新方法的发展，以表征弱蛋白质复合体的三维分子结构。蛋白质-配体之间的强相互作用已被广泛研究，并已确定了许多与生物相关的3D络合物，因为它们的内在稳定性使它们适合于许多分析技术。然而，对于弱蛋白质相互作用，许多传统方法失败或变得不可靠。例如，X射线结晶学，一种非常强大的结构技术，显示了弱相互作用的局限性：(1)获得包括配体在内的配合物的晶体是困难的，(2)描述结合口袋中的配体的电子密度通常定义不佳。核磁共振波谱是研究分子间相互作用的最强大的技术之一，通过使用基于配体的实验，如饱和转移差(STD)核磁共振谱，已经证明了它在检测溶液中弱蛋白质-配体相互作用方面的非凡能力。然而，从这些实验到3D结构的转换目前并不简单。我们已经发表了一些改进的STD核磁共振实验，以确定蛋白质与配体的亲和力，并研究蛋白质结合口袋中配体的多种结合模式。目前的建议源于我们课题组的最新结果，这些结果允许我们提出，与配体接触图或基团表位映射(GEM)相比，STD核磁共振谱可以为弱相互作用提供更多的结构信息。在这里，我们建议获得配体在结合口袋中的取向，这是一个非常需要的信息。我们计划产生新的实验约束来驱动络合物的结构计算，以获得准确的3D结构。到目前为止，这些实验性的限制仍然没有被探索出来。在最具生物相关性的弱相互作用中，蛋白质与多糖的相互作用是许多细胞-细胞交流过程中的关键步骤，也是微生物病原体感染性的关键。特别是，流感病毒利用这一点进行最初的细胞识别、附着和释放新的病毒粒子。在另一种策略中，HIV用宿主多糖覆盖其表面，以逃避免疫反应。有趣的是，新的广谱中和抗体(BNAb)正在被发现，它们能够阻止感染，并针对糖蛋白保护层中的碳水化合物而被激发。在我们与罗布·菲尔德(Norwich)教授和凯蒂·杜尔斯(Katie Doore)博士(伦敦)合作的背景下，我们将应用新的STD核磁共振方法来研究人类和禽流感病毒以及抗HIV的免疫活性bNAbs的分子识别过程。

The extraordinary properties of living systems emerge as a result of dynamic interactions between their biomolecular components (proteins, nucleic acid, carbohydrates or glycans, ...). Despite the complexity of a system of millions of molecular interactions, the whole network of interactions is highly regulated for healthy organisms, and alterations in the regulation processes underlie all diseases. In this network, different molecules interact with different strengths (affinities), where strong interacting partners produce stable biomolecular complexes and weak binding molecules produce transient assemblies. A well "orchestrated" system of protein-ligand interactions of very different affinities is indeed responsible for most of the regulation processes in living organisms. Strong complexes are formed when sustained biological signals are needed, whereas weak interactions are recalled when quick cellular responses are required after temporary stimuli (signal transduction, reversible cell-cell contacts, transient interactions in host/pathogen recognition, etc.). For a complete understanding of life processes, it is necessary to investigate both strong and weak protein-ligand interactions, which encourages the development of novel approaches to characterize the 3D molecular structures of weak protein complexes. Strong protein-ligand interactions have been extensively investigated and many biologically relevant 3D complexes have been determined, as their intrinsic stability makes them amenable to a number of analytical techniques. However, for weak protein interactions many conventional approaches fail or become unreliable. For example, X-ray crystallography, a very powerful structural technique, show limitations for weak interactions as: (i) obtaining crystals of the complexes including the ligand is difficult, and (ii) the typically poorly defined electronic density that describe the ligand in the binding pocket. NMR spectroscopy, one of the most powerful techniques to study intermolecular interactions, has demonstrated its extraordinary capability for the detection of weak protein-ligand interactions in solution, through the use of ligand-based experiments, like Saturation Transfer Difference (STD) NMR spectroscopy. However, the translation from these experiments to 3D structures is currently not straightforward. We have published some improvements in the set up of STD NMR experiments to determine protein-ligand affinities and study multiple binding modes of ligands in a protein binding pocket. The present proposal stems from recent results in our research group that allow us to propose that STD NMR spectroscopy can provide more structural information for weak interactions than the map of ligand contacts, or group epitope mapping (GEM). Here, we propose to obtain the orientation of the ligand in the binding pocket, a much needed piece of information. We plan to generate new experimental restraints to drive the structural calculations of the complexes, to get accurate 3D structures. Until now, these experimental restraints have remained unexplored. Among the most biologically relevant weak interactions, those of proteins with glycans are essential steps in many cell-cell communication processes, and key in the infectivity of microbial pathogens. In particular, influenza virus exploits this for initial cell recognition, attachment, and release of new virions. In a different strategy, HIV covers its surface with host glycans to evade the immunological response. Interestingly, new broad neutralizing antibodies (bNAb) are being discovered which are able to stop infection and are elicited against those carbohydrates in that glycan shield. In the context of our collaborations with Prof. Rob Field (Norwich) and Dr. Katie Doores (London), we will apply the novel STD NMR approaches to investigate the molecular recognition processes in human and avian influenza virus, and in immunologically active bNAbs against HIV.