Oligosaccharide substrate interactions with b-1,4-Galact

糖缀合物的寡糖部分在细胞的几个生物过程中起重要作用，包括糖蛋白的折叠和跨细胞区室的运输。对于这些复杂寡糖的生物合成，细胞中存在复杂的机制。聚糖合成缺陷具有严重的病理后果，并导致几种人类疾病。寡糖部分以高特异性与细胞蛋白结合，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节，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进行定向。分支寡糖交联蛋白质并产生蛋白质-碳水化合物复合物的无限网络，从而调节各种细胞反应。 通过将寡糖对接到结合位点和复合物的MD模拟来定义Gal-T1的寡糖结合位点：我们继续使用分子模拟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，其三维结构最近已在我们的实验室中确定，与UDP-半乳糖和Mn 2+离子复合，或与α-乳白蛋白和N-乙酰葡糖胺复合（见项目编号Z 01 BC 009305-06 LECB）。检查的GlcNAc结合位点在半乳糖-T1从半乳糖-T1 LA GlcNAc晶体结构揭示了一个“开放的运河形”扩展糖结合位点，位于后面的GlcNAc结合位点，平均宽度和长度为10？16？分别该位点由来自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GlcNAc的寡糖如壳二糖竞争。已知有限数量的优选寡糖作为Gal-T1的底物。这些研究表明，在不同的含GlcNAc的二糖中，只有b-连接的二糖如GlcNAcb 1，4-GlcNAc或GlcNAcb 1，2-Man优于a-连接的二糖。事实上，与GlcNAc本身相比，α-甲基-GlcNAc是较不优选的。此外，寡糖如N-聚糖是比（GlcNAc）4更优选的受体底物。与这种N-聚糖连接的蛋白质或肽的存在不影响寡糖的结合。为了探测寡糖结合位点的大小和性质，进行了各种二糖和N-聚糖配体在结合位点中的对接的建模研究。使用CVFF力场和InsightII的Discover模块计算了Gal-T1与糖之间的每个配体构象、分子间相互作用能。能量计算仅考虑距离任何配体原子9 A内的蛋白质残基。通过系统扫描配体的整个立体化学允许区域，在结合位点改变配体的构象。总能量，包括分子间蛋白质-配体相互作用能和分子间配体能量，被用作指导，在确定可能允许的构象的配体在结合位点。这些建模研究表明，具有a-连接取代的GlcNAc如a-苄基-GlcNAc不能结合Gal-T1，因为与高度保守的Tyr 286残基存在严重的空间接触，而具有b-连接取代的GlcNAc如b-苄基-GlcNAc可以在没有任何空间接触的情况下结合。双触角N-聚糖在其还原末端与延伸糖结合位点中的GlcNAc的对接揭示了Gal-T1中的受体结合位点可以从GlcNAc部分一直容纳线性五糖至精氨酸连接的GlcNAc。该结合位点还可以容纳N-聚糖的α-1-3臂（GlcNAc 3b 1 - 2 Man 3a 1 - 3 Manmb 1 - 4GlcNAcb 1 -4GlcNAc-N）或α-1-6臂（GlcNAc 6 b1 - 2 Man 6a 1 - 6 Manmb 1 -4GlcNAcb 1- 4GlcNAc-N），而没有任何空间位阻。MD模拟在水中（75？立方体）显示，在Mana 1 - 3 Manm和GlcNAc 6 b1 - 2 Man 6键的y构象变化在双触角寡糖的结合的1-3-臂中是明显的。在Manmb 1 -4GlcNAc键的y和Man 6a 1 - 6 Manm键的f的构象变化在双触角寡糖的结合1-6臂中是明显的。分子动力学模拟结果表明，当1-3-臂结合在结合位点时，寡糖与Gal-T1之间存在疏水相互作用，从而有利于半乳糖转移到双触角寡糖的1-3-臂上的GlcNAc。这一结论与已发表的生化数据一致。 在人类中，Gal-T1家族成员负责不同寡糖中Gal部分的合成，表明尽管所有这些酶将Gal转移至GlcNAc，但每种酶都识别GlcNAc所连接的剩余寡糖部分不同。人Gal-T家族成员的序列比较揭示了在家族成员中GlcNAc结合位点仅有很少或没有变化，而延伸的寡糖结合区显示出显著的变化，表明这些酶可能偏好不同的含GlcNAc的寡糖作为其优选的糖受体。为了确定寡糖在结合位点中的结合的确切模式，作为第一次尝试，我们已经成功地使Gal-T1突变体与二糖和五糖共结晶。目前正在确定这些配合物的晶体结构。

The oligosaccharide moieties 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 hetrodimerization 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. Defining the oligosaccharide binding site of Gal-T1 by docking oligosaccharides into the binding site and MD simulation of the complexes: 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 three dimensional structure of which has recently been determined in our laboratory, either in complex with UDP-galactose and Mn2+ion, or in complex with a-lactalbumin and N-acetylglucosamine (see Project # Z01 BC 009305-06 LECB). 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 ? and 16 ?, 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. A limited number of preferred oligosaccharides as substrates for Gal-T1 are known. These studies have shown that among the different GlcNAc containing disaccharides, only a b-linked disaccharide such as GlcNAcb1,4-GlcNAc or GlcNAcb1,2-Man is 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. The presence of the protein or peptide attached to such an N-glycan does not influence the binding of an oligosaccharide. In order to probe the size and nature of the oligosaccharide binding site, a modeling study of the docking of various disaccharides and N-glycan ligands in the binding site were carried out. Each ligand conformation, inter-molecular interaction energy between Gal-T1 and the saccharide was calculated using CVFF force field and Discover module of InsightII. Only protein residues within 9 A from any of the ligand atoms were considered for energy calculations. The conformation of the ligand was varied in the binding site by systematically scanning the entire stereochemically allowed region of the ligand. The total energy, comprising the intermolecular protein-ligand interaction energy and intermolecular ligand energy, was used as a guide in determining the possible allowed conformations of the ligand in the binding site. These modeling studies show that GlcNAc with an a-linked substitution such as a-benzyl-GlcNAc can not bind to Gal-T1 because of severe steric contacts with the highly conserved Tyr286 residue, whereas GlcNAc with a b-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 (GlcNAc3b1-2Man3a1-3Manmb1-4GlcNAcb1-4GlcNAc-N) or a-1-6 arm (GlcNAc6b1-2Man6a1-6Manmb1-4GlcNAcb 1-4GlcNAc-N) of the N-glycan without any steric hindrance. MD simulations in water (75 ? cube) for 750 ps of Gal-T1 alone or with the docked bi-antennary oligosaccharide in the binding site show that the conformational changes in the y at the Mana1-3Manm and GlcNAc6b1-2Man6 linkage are pronounced in the bound 1-3-arm of the bi-antennary oligosaccharide. Conformational changes in y at the Manmb1-4GlcNAc linkage, and f at the Man6a1-6Manm linkage are pronounced in the bound 1-6-arm of the bi-antennary oligosaccharide. The MD simulation results suggest hydrophobic interactions between the oligosaccharide and Gal-T1 when 1-3-arm is bound in the binding site, thus favoring the transfer of galactose to the GlcNAc on the 1-3-arm of the bi-antennary oligosaccharide. This conclusion concurs with the published biochemical data. In humans Gal-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 Gal-T family members reveals only a little or no variation in the GlcNAc binding site among the family members 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, as a first attempt we have been successful in co-crystallizing Gal-T1 mutants with di and a penta-saccharide. The crystal structure of these complexes is currently being determined.