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

糖偶联物的寡糖部分在细胞的几个生物学过程中发挥着重要作用，包括糖蛋白的折叠和跨细胞间隔的运输。低聚糖以高度的特异性与细胞蛋白结合，调节糖蛋白的同源和异构化。由于低聚糖的构象灵活性，二糖单元的扭转角，特别是围绕A1-6-键的扭转角，以一种方式调整，使寡糖的侧基以促进与蛋白质结合残基的有利相互作用的方式自我定位。分枝低聚糖使蛋白质交联，并产生无限的蛋白质-碳水化合物复合体网络，从而调节各种细胞反应。糖链合成缺陷已被证明具有严重的病理后果，并导致几种人类疾病。 通过将寡糖对接到结合位点来确定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，尤其是各种寡糖底物与Gal-T1的结合，最近我们实验室已经确定了Gal-T1的三维结构，无论是与UDP-半乳糖和金属离子的络合物，还是与a-乳蛋白和N-乙酰氨基葡萄糖的络合物(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。在不同的含二糖的GlcNAc中，只有b连接的二糖如GlcNAcb1，4-GlcNAc或GlcNAcb1，2-Man优先于a连接的二糖。事实上，与GlcNAc本身相比，a-甲基-GlcNAc不那么受欢迎。此外，N-聚糖等寡糖是比a(GlcNAc)4更好的受体底物。从Gal-T1-LA-GlcNAc晶体结构中检测Gal-T1中的GlcNAc结合部位，发现在GlcNAc结合部位后面有一个“开渠状”的延伸糖结合部位，平均宽度和长度分别为10A和16A。这个位点由三个区域的残基组成：残基280到289，残基319到325和残基359到368。在Gal-T1-LA络合物的晶体结构中，La与这一区域结合，因此有望与含有壳二糖等寡糖的GlcNAc竞争。为了了解Gal-T1中寡糖结合位点的大小和性质，我们使用各种二糖和N-糖链对接到Gal-T1的结合部位，而没有在对接过程中使任何一个分子的能量最小化。这些研究表明，具有a-取代的GlcNAc不能与Gal-T1结合，因为它的结合与高度保守的残基Tyr286产生了严重的空间接触，而具有a-取代的GlcNAc例如b-苄基-GlcNAc可以在没有任何空间接触的情况下结合。二天线N-葡聚糖与扩展糖结合位点上的GlcNAcs的对接表明，Gal-T1中的受体结合位点可以容纳从GlcNAc部分到天冬氨酸连接的GlcNAc的线性五糖。结合部位也可以容纳N-糖链的a-1-3臂(GlcNAcb1-2Mana1-3Man b1-4GlcNAcb1-4GlcNAc-N)或-1-6臂(GlcNAcb1-2Mana1-6Manb1-4GlcNAcb1-4GlcNAc-N)，而不存在任何空间位阻。 在人类中，Gal-T1家族成员负责合成不同寡糖中的Gal部分，这表明这些酶虽然将Gal转移到GlcNAc，但识别GlcNAc连接的剩余寡糖部分。因此，定义在牛Gal-T1上的寡糖结合位点对于了解人类Gal-T家族成员的结构和功能具有重要意义。家族成员的GlcNAc结合位点的序列比较发现，序列变化很小或没有变化，而延伸的寡糖结合区显示出显著的差异，这表明这些酶可能更喜欢不同的含低聚糖的GlcNAc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他们偏好的确切性质正在进行中。 1)Qasba PK。碳水化合物聚合物41,293-309,2000年。 Z01 BC 10041-04