A Simple Method for Discovering Druggable, Specific Glycosaminoglycan-Protein Systems. Elucidation of Key Principles from Heparin/Heparan Sulfate-Binding Proteins

Glycosaminoglycans (GAGs) affect human physiology and pathology by modulating more than 500 proteins. GAG-protein interactions are generally assumed to be ionic and nonspecific, but specific interactions do exist. Here, we present a simple method to identify the GAG-binding site (GBS) on proteins that in turn helps predict high specific GAG–protein systems. Contrary to contemporary thinking, we found that the electrostatic potential at basic arginine and lysine residues neither identifies the GBS consistently, nor its specificity. GBSs are better identified by considering the potential at neutral hydrogen bond donors such as asparagine or glutamine sidechains. Our studies also reveal that an unusual constellation of ionic and non-ionic residues in the binding site leads to specificity. Nature engineers the local environment of Asn45 of antithrombin, Gln255 of 3-O-sulfotransferase 3, Gln163 and Asn167 of 3-O-sulfotransferase 1 and Asn27 of basic fibroblast growth factor in the respective GBSs to induce specificity. Such residues are distinct from other uncharged residues on the same protein structure in possessing a significantly higher electrostatic potential, resultant from the local topology. In contrast, uncharged residues on nonspecific GBSs such as thrombin and serum albumin possess a diffuse spread of electrostatic potential. Our findings also contradict the paradigm that GAG-binding sites are simply a collection of contiguous Arg/Lys residues. Our work demonstrates the basis for discovering specifically interacting and druggable GAG-protein systems based on the structure of protein alone, without requiring access to any structure-function relationship data.糖胺聚糖（Glycosaminoglycans, GAGs）通过调控五百余种蛋白质，影响人体的生理与病理过程。学界通常认为GAG与蛋白质的相互作用属于离子型相互作用且不具备特异性，但实际上特异性的GAG-蛋白质相互作用确实存在。我们提出一种简便方法，可识别蛋白质上的糖胺聚糖结合位点（GAG-binding site, GBS），该方法能够助力预测高特异性的GAG-蛋白质相互作用体系。与当前主流认知相悖的是，我们发现碱性精氨酸与赖氨酸残基处的静电电势，既无法稳定识别糖胺聚糖结合位点，也无法判断其结合特异性。相较而言，通过分析天冬酰胺或谷氨酰胺侧链这类中性氢键供体处的静电电势，可更精准地识别糖胺聚糖结合位点。我们的研究还揭示，结合位点内离子与非离子残基的特殊排布，是赋予结合特异性的关键。大自然通过改造抗凝血酶的Asn45、3-O-磺基转移酶3（3-O-sulfotransferase 3）的Gln255、3-O-磺基转移酶1（3-O-sulfotransferase 1）的Gln163与Asn167，以及碱性成纤维细胞生长因子的Asn27在各自糖胺聚糖结合位点中的局部环境，以此诱导产生结合特异性。这类残基与同一蛋白质结构上的其他非带电残基存在显著差异：它们具备显著更高的静电电势，这一特性源于其局部拓扑结构。与之形成鲜明对比的是，凝血酶、血清白蛋白这类非特异性GAG结合位点上的非带电残基，其静电电势分布较为弥散。我们的研究结果同样驳斥了"糖胺聚糖结合位点仅为连续精氨酸/赖氨酸残基集合"这一传统范式。本研究证明，仅依托蛋白质结构，即可发现具有特异性相互作用且可成药的GAG-蛋白质相互作用体系，无需借助任何结构-功能关系数据。