Many host pathogen interactions such as human viruses (including non-SARS-coronaviruses) rely on attachment to host cell-surface glycans. There are conflicting reports about whether the Spike protein of SARS-CoV-2 binds to sialic acid commonly found on host cell-surface N-linked glycans. In the absence of a biochemical assay, the ability to analyze the binding of glycans to heavily- modified proteins and resolve this issue is limited. Classical Saturation Transfer Difference (STD) NMR can be confounded by overlapping sugar resonances that compound with known experimental constraints. Here we present ‘universal saturation transfer analysis’ (uSTA), an NMR method that builds on existing approaches to provide a general and automated workflow for studying protein-ligand interactions. uSTA reveals that B-origin-lineage-SARS-CoV-2 spike trimer binds sialoside sugars in an ‘end on’ manner and modelling guided by uSTA localises binding to the spike N-terminal domain (NTD). The sialylated-polylactosamine motif is found on tetraantennary human N-linked-glycoproteins in deeper lung and may have played a role in zoonosis. Provocatively, sialic acid binding is abolished by mutations in some subsequent SARS- CoV-2 variants-of-concern. A very high resolution cryo-EM structure confirms the NTD location and ‘end on’ mode; it rationalises the effect of NTD mutations and the structure-activity relationship of sialic acid analogues. uSTA is demonstrated to be a robust, rapid and quantitative tool for analysis of binding, even in the most demanding systems.

中文翻译：

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通用饱和转移分析揭示的隐秘病原体-糖相互作用

许多宿主病原体相互作用，例如人类病毒（包括非 SARS 冠状病毒）依赖于宿主细胞表面聚糖的附着。关于 SARS-CoV-2 的 Spike 蛋白是否与宿主细胞表面 N-连接聚糖上常见的唾液酸结合，存在相互矛盾的报道。在没有生化分析的情况下，分析聚糖与高度修饰的蛋白质的结合并解决这个问题的能力是有限的。经典的饱和转移差 (STD) NMR 可能会被重叠的糖共振混淆，这些共振与已知的实验限制相结合。在这里，我们提出了“通用饱和转移分析”（uSTA），这是一种 NMR 方法，它建立在现有方法的基础上，为研究蛋白质-配体相互作用提供通用和自动化的工作流程。uSTA 揭示了 B-origin-lineage-SARS-CoV-2 尖峰三聚体以“end on”方式结合唾液酸糖，并且由 uSTA 指导的建模将与尖峰 N 末端结构域 (NTD) 的结合定位。唾液酸化聚乳糖胺基序存在于更深肺中的四触角人类 N 连接糖蛋白上，可能在人畜共患病中发挥作用。具有挑战性的是，唾液酸结合被一些随后的 SARS-CoV-2 关注变体中的突变消除。非常高分辨率的冷冻电镜结构确认了 NTD 位置和“结束”模式；它使 NTD 突变的影响和唾液酸类似物的构效关系合理化。uSTA 被证明是一种强大、快速和定量的结合分析工具，即使在最苛刻的系统中也是如此。