Center of Structural and Cell Biology in Medicine, Institute of Chemistry and Metabolomics, University of Luebeck, Ratzeburger Allee 160, 23562, Luebeck, Germany.
Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland.
Glycoconj J. 2017 Oct;34(5):679-689. doi: 10.1007/s10719-017-9792-5. Epub 2017 Aug 19.
Attachment of human noroviruses to histo blood group antigens (HBGAs) is thought to be critical for the infection process. Therefore, we have determined binding epitopes of synthetic type 1 to 6 blood group A- and B-tetrasaccharides binding to GII.4 human Norovirus virus like particles (VLPs) using STD NMR experiments. So far, little information is available from crystal structure analysis studies on the interactions of the reducing-end sugars with the protruding domain (P-domain) of the viral coat protein VP1. Here, we show that the reducing-end sugars make notable contacts with the protein surface. The type of glycosidic linkage, and the identity of the sugar at the reducing end modulate HBGA recognition. Most strikingly, type 2 structures yield only very poor saturation transfer indicating impeded binding. This observation is in accordance with previous mass spectrometry based affinity measurements, and can be understood based on recent crystal structure data of a complex of highly homologous GII.4 P-dimers with H-type 2 trisaccharide where the N-acetyl group of the reducing N-acetyl glucosamine residue points towards a loop comprising amino acids Q390 to H395. We suggest that in our case, binding of type 2 A- and B-tetrasaccharides leads to steric conflicts with this loop. In order to identify factors determining L-Fuc recognition, we also synthesized GII.4 VLPs with point mutations D391A and H395A. Prior studies had suggested that these residues, located in a second shell around the L-Fuc binding site, assist L-Fuc binding. STD NMR experiments with L-Fuc and B-trisaccharide in the presence of wild type and mutant VLPs yield virtually identical binding epitopes suggesting that these two mutations do not significantly alter HBGA recognition. Our study emphasizes that recognition of α-(1→2)-linked L-Fuc residues is a conserved feature of GII.4 noroviruses. However, structural variation of the HBGA core structures clearly modulates molecular recognition depending on the genotype.
人诺如病毒与组织血型抗原（HBGA）的附着被认为对感染过程至关重要。因此，我们使用 STD NMR 实验确定了与 GII.4 人类诺如病毒病毒样颗粒（VLPs）结合的合成型 1 至 6 型 A 和 B 四糖结合物的结合表位。到目前为止，从晶体结构分析研究中获得的有关还原端糖与病毒衣壳蛋白 VP1 的突出结构域（P 结构域）相互作用的信息很少。在这里，我们表明还原端糖与蛋白质表面有明显的接触。糖苷键的类型以及还原端糖的种类都调节 HBGA 的识别。最引人注目的是，2 型结构仅产生非常差的饱和转移，表明结合受到阻碍。这一观察结果与之前基于质谱的亲和力测量结果一致，并且可以根据最近高度同源的 GII.4 P-二聚体与 H 型 2 三糖复合物的晶体结构数据来理解，其中还原 N-乙酰葡萄糖胺残基的 N-乙酰基的 N-乙酰基指向包含氨基酸 Q390 至 H395 的环。我们认为，在我们的情况下，2 型 A 和 B 四糖的结合会导致与该环的空间位阻冲突。为了确定决定 L-Fuc 识别的因素，我们还合成了具有点突变 D391A 和 H395A 的 GII.4 VLPs。先前的研究表明，这些位于 L-Fuc 结合位点周围的第二壳层中的残基，有助于 L-Fuc 结合。在存在野生型和突变型 VLPs 的情况下，使用 L-Fuc 和 B-三糖进行 STD NMR 实验得到了几乎相同的结合表位，这表明这两个突变不会显著改变 HBGA 的识别。我们的研究强调，α-（1→2）-连接的 L-Fuc 残基的识别是 GII.4 诺如病毒的一个保守特征。然而，HBGA 核心结构的结构变异显然会根据基因型调节分子识别。
