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

糖缀合物的寡糖部分在细胞的几个生物过程中起着重要作用，包括糖蛋白在跨细胞室中的折叠和转运。寡糖部分与具有高特异性的细胞蛋白结合，并调节糖蛋白的同型和乙酸二聚体。由于寡糖的构象柔韧性，二糖单元的扭转角，尤其是在A1-6-链接周围，以这样的方式进行调整，以至于寡糖的侧基团体以一种促进与蛋白质结合残基的有利相互作用的方式进行了良好的形式。分支的寡糖交联蛋白，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。有缺陷的聚糖合成已显示出严重的病理后果，并导致几种人类疾病。 通过将寡糖对接到结合位点中的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与蛋白质的结合，特别是在各种寡糖均与蛋白质之间的结合，在我们的3d结构中，该工具均与gal-the的结合，在gal-the的各种结合中，在gal-the中的结合，是gal-t的结合。与UDP-半乳糖和锰金属离子，或与A-乳蛋白和N-乙酰基葡萄糖胺复合体（请参阅Project＃Z01 BC 009304-05 LECB）。数量有限的寡糖已显示为GAL-T1的首选底物。在含有二糖的不同GLCNAC中，仅是B连接的二糖，例如GlcNACB1,4-GLCNAC或GLCNACB1,2-MAN优先于A连接的二糖。实际上，与GlcNAC本身相比，A-甲基-GLCNAC比GlcNAC更不受欢迎。同样，诸如N-聚糖之类的寡糖比A（GlcNAC）4更优选的受体底物。从GAL-T1-LA-LA-GLCNAC晶体结构中检查GAL-T1中的GlcNAC结合位点表明，GlcNAC结合位点后面的“开放管形”延长的糖结合位点，平均宽度和长度为10 a和16 a。该地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与该区域结合Gal-T1-LA复合物的晶体结构，因此预计它将与含有寡糖的GlcNAC竞争。为了理解GAL-T1中寡糖结合位点的大小和性质，我们已经使用各种二糖和N-聚糖进行了建模研究，以将其扩展到GAL-T1的结合位点，而在不可能最小化对接过程中的任何一个分子的情况下。这些研究表明，具有A链取代的GlcNAC（例如A-苯基GlCNAC）无法与GAL-T1结合，因为其结合产生了与高度保守的残基Tyr286的严重空间接触，而作为GlcNAC，GlcNAC具有与benzyl-glcnac的链接替代物，可以在没有任何空间的情况下结合。在扩展的糖结合位点，在其还原末端与glcnacs的双胞质N-聚糖对接，gal-T1中的受体结合位点可以容纳从GlcNAC部分到芦笋链接的GlcNAC的线性五糖。结合位点还可以容纳A-1-3 ARM（GLCNACB1-3MAN B1-4GLCNACB1-4GLCNAC-N）或A-1-6 ARM（GLCNACB1-2MANA1-6MANB1-6MANB1-4GLCNACB 1-4GLCNAC-N），没有任何n-n-n-n-Glyculance。 在人类中，gal-t1家族成员在不同的寡糖中合成gal部分的合成，表明这些酶虽然将GAL转移到GlcNAC中，但却识别出剩余的GlcNAC附着在其上的寡糖部分。因此，在牛gal-t1上定义的寡糖结合位点对于理解人gal-t家族成员的结构功能很重要。家族成员的GlcNAC结合位点的序列比较显示出一点序列变化，而扩展的寡糖结合区域则显示出显着的变化，表明这些酶可能更喜欢含有寡糖作为其首选糖受体的不同GlcNAC。但是，基于同源性建模及其晶体结构确定的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。 Z01 BC 10041-04