Oligosaccharide Interactions with Proteins

低聚糖与蛋白质的相互作用

• 批准号: 6559116
• 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/6559116
• 关键词: binding proteins; chemical conjugate; chemical structure function; computer simulation; conformation; enzyme substrate complex; galactosyltransferases; glycoproteins; intermolecular interaction; model design /development; molecular dynamics; molecular site; oligosaccharides; physical model; protein metabolism; 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. 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 a1-6-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. Defective glycan synthesis has been shown to have serious pathological consequences and result in several human diseases. Defining the oligosaccharide binding site of b-1,4-Galactosyltransferase(Gal-T1) by docking of oligosaccharides into the binding site: 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 Gal-T1, the 3D-structure of which has recently been determined in our laboratory, either in complex with UDP-galactose and manganese metal ion, or in complex with a-lactalbumin and N-acetylglucosamine (see Project # Z01 BC 009304-05 LECB ). A limited number of oligosaccharides have been shown to be the preferred substrates for Gal-T1. Among the different GlcNAc containing disaccharides only a b-linked disaccharide such as GlcNAcb1,4-GlcNAc or GlcNAcb1,2-Man are preferred over a-linked disaccharides. In fact a-methyl-GlcNAc is less preferred compared to GlcNAc by itself. Also, oligosaccharides such as N-glycans are more preferred acceptor substrates than a (GlcNAc)4. Examination of the GlcNAc binding site in Gal-T1 from the Gal-T1-LA-GlcNAc crystal structure reveals an "open canal shaped" extended sugar binding site that lies behind the GlcNAc binding site, with an average width and length of 10 A and 16 A, respectively. 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 Gal-T1-LA complex, therefore it is expected to compete with the GlcNAc containing oligosaccharides such as chitobiose. In order to understand the size and nature of the oligosaccharide binding site in Gal-T1 we have carried out a modeling study using various disaccharides and N-glycans to dock into the binding site of Gal-T1 without energy minimization of either of the molecules during docking. These studies indicate that GlcNAc with an a-linked substitution such as a-benzyl-GlcNAc can not binding to Gal-T1 since its binding creates a severe steric contacts with the highly conserved residue Tyr286, while as GlcNAc with a -linked substitution such as b-benzyl-GlcNAc can bind without any steric contacts. Docking of a biantennary N-glycan with GlcNAcs at its reducing ends in the extended sugar binding site reveals that the acceptor binding site in Gal-T1 can accommodate a linear pentasaccharide all the way from the GlcNAc moiety to the aspargine linked GlcNAc. The binding site can also accommodate either the a-1-3 arm (GlcNAcb1-2Mana1-3Man b1-4GlcNAcb1-4GlcNAc-N) or a-1-6 arm (GlcNAcb1-2Mana1-6Manb1-4GlcNAcb 1-4GlcNAc-N) of the N-glycan without any steric hindrance. In humans Gal-T1 family members are responsible for the synthesis of Gal moiety in different oligosaccharides, indicating that these enzymes, although transfer Gal to GlcNAc, recognize the remaining oligosaccharide moieties to which GlcNAc is attached to. Therefore the oligosaccharide binding site defined on bovine Gal-T1 will be important in understanding the structure-function of human Gal-T family members. The sequence comparison of the GlcNAc binding site of the family members reveals a little or no sequence variation, while the extended oligosaccharide binding region shows significant variations, indicating that these enzymes may prefer different GlcNAc containing oligosaccharides as their preferred sugar acceptors. However, the exact nature of their preferences based on the homology modeling and their crystal structure determination is underway. 1) Qasba PK. Carbohydrate Polymers 41, 293-309, 2000. Z01 BC 10041-04

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。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 a1-6-键的扭转角，以这样的方式进行调整，使得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。聚糖合成缺陷已被证明会产生严重的病理后果并导致多种人类疾病。 通过将寡糖对接到结合位点来定义b-1,4-半乳糖基转移酶（Gal-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Gal-T1的结合，其3D结构最近已在我们的实验室中确定，无论是在复杂的 与 UDP-半乳糖和锰金属离子，或与 a-乳清蛋白和 N-乙酰葡糖胺复合（参见项目# Z01 BC 009304-05 LECB）。有限数量的寡糖已被证明是 Gal-T1 的首选底物。在不同的含有GlcNAc的二糖中，仅b-连接的二糖例如GlcNAcb1,4-GlcNAc或GlcNAcb1,2-Man比α-连接的二糖优选。事实上，与GlcNAc本身相比，α-甲基-GlcNAc不太优选。而且，寡糖例如N-聚糖是比(GlcNAc) 4 更优选的受体底物。从 Gal-T1-LA-GlcNAc 晶体结构检查 Gal-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，平均宽度和长度分别为 10 A 和 16 A。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Gal-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。为了了解 Gal-T1 中寡糖结合位点的大小和性质，我们进行了一项建模研究，使用各种二糖和 N-聚糖对接到 Gal-T1 的结合位点，而在对接过程中没有任何一个分子的能量最小化。这些研究表明，具有α-连接取代（例如α-苄基-GlcNAc）的GlcNAc不能与Gal-T1结合，因为其结合与高度保守的残基Tyr286产生严格的空间接触，而具有α-连接取代（例如b-苄基-GlcNAc）的GlcNAc可以在没有任何空间接触的情况下结合。双触角 N-聚糖在延伸的糖结合位点的还原端与 GlcNAc 对接表明，Gal-T1 中的受体结合位点可以容纳从 GlcNAc 部分到天冬酰胺连接的 GlcNAc 的线性五糖。结合位点还可以容纳 N-聚糖的 a-1-3 臂 (GlcNAcb1-2Mana1-3Man b1-4GlcNAcb1-4GlcNAc-N) 或 a-1-6 臂 (GlcNAcb1-2Mana1-6Manb1-4GlcNAcb 1-4GlcNAc-N)，而没有任何空间位阻。 在人类中，Gal-T1 家族成员负责不同寡糖中 Gal 部分的合成，表明这些酶虽然将 Gal 转移到 GlcNAc，但仍识别 GlcNAc 所附着的其余寡糖部分。因此，牛 Gal-T1 上定义的寡糖结合位点对于理解人类 Gal-T 家族成员的结构功能非常重要。家族成员的 GlcNAc 结合位点的序列比较显示出很少或没有序列变化，而延伸的寡糖结合区显示出显着的变化，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。然而，基于同源建模和晶体结构确定的其偏好的确切性质正在进行中。 1）卡斯巴PK。碳水化合物聚合物 41, 293-309, 2000。 Z01 BC 10041-04