Schilling Philipp E, Kontaxis Georg, Dragosits Martin, Schiestl Robert H, Becker Christian F W, Maier Irene
Faculty of Chemistry, Institute of Biological Chemistry, University of Vienna Währinger Straße 38 A-1090 Vienna Austria.
Department of Structural and Computational Biology, Max Perutz Laboratories, University of Vienna Campus Vienna Bohrgasse 5 A-1030 Vienna Austria.
RSC Adv. 2020 Mar 17;10(19):11079-11087. doi: 10.1039/d0ra01128b. eCollection 2020 Mar 16.
Cyanovirin-N (CV-N) has been shown to reveal broad neutralizing activity against human immunodeficiency virus (HIV) and to specifically bind Manα(1→2)Manα units exposed on various glycoproteins of enveloped viruses, such as influenza hemagglutinin (HA) and Ebola glycoprotein. Chemically synthesized dimannosylated HA peptides bound domain-swapped and dimeric CV-N with either four disulfide-bonds (Cys-Cys), or three Cys-Cys bonds and an intact fold of the high-affinity binding site at an equilibrium dissociation constant of 10 μM. Cys-Cys mutagenesis with ion-pairing amino-acids glutamic acid and arginine was calculated by structure-based protein design and allowed for recognizing dimannose and dimannosylated peptide binding to low-affinity binding sites ( ≈ 11 μM for one C58-C73 bond, and binding to dimannosylated peptide). In comparison, binding to HA was achieved based on one ion-pairing C58E-C73R substitution at = 275 nM, and = 5 μM for two C58E-C73R substitutions. We were utilizing a triazole bioisostere linkage to form the respective mannosylated-derivative on the HA peptide sequence of residues glutamine, glycine, and glutamic acid. Thus, mono- and dimannosylated peptides with N-terminal cysteine facilitated site-specific interactions with HA peptides, mimicking a naturally found N-linked glycosylation site on the HA head domain.
蓝绿菌素-N（CV-N）已被证明对人类免疫缺陷病毒（HIV）具有广泛的中和活性，并能特异性结合包膜病毒各种糖蛋白上暴露的Manα(1→2)Manα单元，如流感血凝素（HA）和埃博拉糖蛋白。化学合成的二甘露糖基化HA肽与结构域交换的二聚体CV-N结合，该CV-N具有四个二硫键（Cys-Cys），或三个Cys-Cys键以及高亲和力结合位点的完整折叠，平衡解离常数为10 μM。通过基于结构的蛋白质设计计算了带有离子配对氨基酸谷氨酸和精氨酸的Cys-Cys诱变，从而识别二甘露糖和二甘露糖基化肽与低亲和力结合位点的结合（对于一个C58-C73键，Kd≈11 μM，以及与二甘露糖基化肽的结合）。相比之下，基于一个离子配对的C58E-C73R取代，在Kd = 275 nM时实现了与HA的结合，对于两个C58E-C73R取代，Kd = 5 μM。我们利用三唑生物电子等排体连接在谷氨酰胺、甘氨酸和谷氨酸残基的HA肽序列上形成相应的甘露糖基化衍生物。因此，具有N端半胱氨酸的单甘露糖基化和二甘露糖基化肽促进了与HA肽的位点特异性相互作用，模拟了HA头部结构域上天然存在的N-连接糖基化位点。
