Oligosaccharide substrate interactions with beta-1,4-Ga

寡糖底物与 beta-1,4-Ga 的相互作用

• 批准号: 6944635
• 负责人: Pradman K Qasba
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6944635
• 关键词: N acetylglucosamine; binding sites; carbohydrate biosynthesis; carbohydrate structure; chemical models; computer simulation; conformation; crystallization; enzyme mechanism; enzyme structure; enzyme substrate complex; galactose; galactosyltransferases; glycoproteins; intermolecular interaction; molecular dynamics; oligosaccharides; protein binding; structural biology

The oligosaccharide moiety of glycoconjugates play important roles in several biological processes of a cell, including the folding and transport of glycoproteins across cellular compartments. For the biosynthesis of these complex oligosaccharides an intricate machinery exists in a cell. Defective glycan synthesis has serious pathological consequences and results in several human diseases. The oligosaccharide moieties bind to cellular proteins with high specificity and modulate the homo- and hetro-dimerization of glycoproteins. Due to the conformational flexibility of oligosaccharides, the torsional angles of a disaccharide unit, especially around the alpha-1-6-linkage, adjust in such a way that the side groups of the oligosaccharides orient themselves in a manner that promotes favorable interactions with the binding residues of the protein. Branched oligosaccharides cross-link proteins and generate infinite networks of protein-carbohydrate complexes, resulting in the modulation of various cell responses. In humans b4Gal-T1 family members are responsible for the synthesis of Gal moiety in different oligosaccharides, indicating that although all these enzymes transfer Gal to GlcNAc, each recognizes the remaining oligosaccharide moieties to which GlcNAc is attached differently. The sequence comparison of the human b4Gal-T family members reveal only a little or no variation among the family members in the GlcNAc binding site where as the extended oligosaccharide binding region shows significant variations, indicating that these enzymes may prefer different GlcNAc containing oligosaccharides as their preferred sugar acceptors. To determine the exact mode of binding of the oligosaccharide in the binding site we have carried both MD simulations as well as crystal structure analysis of the b4Gal-T1-oligosaccharide complexes. Defining the oligosaccharide binding site of b4Gal-T1 by docking oligosaccharides into the binding site and by crystal structure investigation of the complexes with the oligosaccharides : We have continued to use molecular modeling methods to study the binding of oligosaccharides to proteins, in particular the binding of various oligosaccharide substrates to b4Gal-T1, the 3D-structure of which has been determined in our laboratory, either in complex with UDP-galactose and Mn2+ ion, or in complex with alpha-lactalbumin and N-acetylglucosamine (see Project # Z01 BC 09305-08 LECB). Examination of the GlcNAc binding site in b4Gal-T1 from the crystal structure reveals an "open canal shaped" extended sugar binding site that lies behind the GlcNAc binding site. This site is formed by the residues from three regions; residues 280 to 289, residues 319 to 325 and residues 359 to 368. LA binds to this region in the crystal structure of b4Gal-T1-LA complex, therefore it is expected to compete with the GlcNAc containing oligosaccharides such as chitobiose. These modeling studies have shown, which have concurred that among the different GlcNAc containing disaccharides only beta-linked disaccharides such as GlcNAc-beta-1,4-GlcNAc or GlcNAc-beta-1,2-Man are preferred over alpha-linked disaccharides. In fact alpha-methyl-GlcNAc is less preferred compared to GlcNAc by itself. Crystallization of the wild type b4Gal-T1with the acceptor either in the presence or absence of UDP has not been successful so far. This is mainly due to the absence of the acceptor binding-site in the apo-b4Gal-T1 that exists in the open conformation. The enzyme has been crystallized in the closed conformation, where the acceptor site is present, only when UDP-Gal is bound. Although UDP or the acceptor molecules can induce the essential conformational changes, such complexes have been crystallized thus far only in the presence of LA. Since LA binds to the extended sugar binding site it is not possible to crystallize b4Gal-T1 with the oligosaccharide acceptors in the presence of LA.

糖复合物的寡糖部分在细胞的多个生物过程中发挥重要作用，包括糖蛋白跨细胞区室的折叠和运输。对于这些复杂寡糖的生物合成，细胞中存在着复杂的机制。聚糖合成缺陷会产生严重的病理后果，并导致多种人类疾病。寡糖部分以高特异性结合细胞蛋白质并调节糖蛋白的同源和异源二聚化。由于寡糖的构象灵活性，二糖单元的扭转角，特别是围绕 α-1-6-键的扭转角，以这样的方式进行调整，即寡糖的侧基以促进与蛋白质的结合残基的有利相互作用的方式自行定向。支链寡糖交联蛋白质并产生无限的蛋白质-碳水化合物复合物网络，从而调节各种细胞反应。 在人类中，b4Gal-T1 家族成员负责不同寡糖中 Gal 部分的合成，这表明尽管所有这些酶都将 Gal 转移到 GlcNAc，但每种酶对 GlcNAc 附着的其余寡糖部分的识别方式不同。人类 b4Gal-T 家族成员的序列比较揭示了家族成员之间在 GlcNAc 结合位点上仅有很小的变化或没有变化，而延伸的寡糖结合区域显示出显着的变化，表明这些酶可能更喜欢含有不同 GlcNAc 的寡糖作为它们的首选糖受体。为了确定寡糖在结合位点的确切结合模式，我们对 b4Gal-T1-寡糖复合物进行了 MD 模拟和晶体结构分析。 通过将寡糖对接至结合位点并研究寡糖复合物的晶体结构来定义 b4Gal-T1 的寡糖结合位点：我们继续使用分子建模方法来研究寡糖与蛋白质的结合，特别是各种寡糖底物与 b4Gal-T1 的结合，其 3D 结构已确定 在我们的实验室中，要么与 UDP-半乳糖和 Mn2+ 离子形成复合物，要么与 α-乳清蛋白和 N-乙酰葡糖胺形成复合物（参见项目 # Z01 BC 09305-08 LECB）。从晶体结构检查 b4Gal-T1 中的 GlcNAc 结合位点揭示了位于 GlcNAc 结合位点后面的“开放运河形”延伸糖结合位点。该位点由三个区域的残基形成；残基280至289、残基319至325和残基359至368。LA与b4Gal-T1-LA复合物晶体结构中的该区域结合，因此预期其与含有寡糖(例如壳二糖)的GlcNAc竞争。这些模型研究表明，在不同的含有GlcNAc的二糖中，只有β-连接的二糖例如GlcNAc-β-1,4-GlcNAc或GlcNAc-β-1,2-Man比α-连接的二糖更优选。事实上，与 GlcNAc 本身相比，α-甲基-GlcNAc 不太优选。迄今为止，无论存在或不存在UDP，野生型b4Gal-T1与受体的结晶尚未成功。这主要是由于开放构象中存在的apo-b4Gal-T1 中缺乏受体结合位点。仅当 UDP-Gal 结合时，酶才会以闭合构象结晶，其中存在受体位点。尽管 UDP 或受体分子可以诱导必要的构象变化，但迄今为止，此类复合物仅在 LA 存在的情况下才会结晶。由于 LA 与延伸的糖结合位点结合，因此在 LA 存在的情况下不可能使 b4Gal-T1 与寡糖受体结晶。