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。对于这些复杂的低聚糖的生物合成，细胞中存在一种复杂的机制。糖链合成缺陷会造成严重的病理后果，并导致多种人类疾病。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α-1-6-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。 在人类中，b4Gal-T1家族成员负责合成不同寡糖中的Gal部分，这表明虽然所有这些酶都将Gal转移到GlcNAc，但每个成员都识别GlcNAc所连接的剩余寡糖部分。人类b4Gal-T家族成员的序列比较发现，在GlcNAc结合位点上的家族成员之间只有很小的差异或没有差异，而在延伸的寡糖结合区显示出显著的差异，这表明这些酶可能偏好不同的含有寡糖的GlcNAc作为它们的首选糖受体。为了确定低聚糖在结合部位的确切结合方式，我们对B4Gal-T1-寡糖络合物进行了MD模拟和晶体结构分析。 通过将低聚糖对接到结合位点并通过对低聚糖与低聚糖配合物的晶体结构的研究来确定b4Gal-T1的低聚糖结合部位：我们继续使用分子模拟方法来研究低聚糖与蛋白质的结合，尤其是各种寡糖底物与b4Gal-T1的结合，我们的实验室已经确定了B4Gal-T1的三维结构，无论是与UDP-半乳糖和Mn2+形成的络合物，还是与α-乳蛋白和N-乙酰氨基葡萄糖形成的络合物(见项目#Z01 BC 09305-08 LECB)。从晶体结构中检查B4Gal-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。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B4Gal-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。这些模拟研究表明，在不同的含有二糖的GlcNAc中，只有β-连接的二糖，如GlcNAc-β-1，4-GlcNAc或GlcNAc-β-1，2-Man比α连接的二糖更受欢迎。事实上，与GlcNAc本身相比，α-甲基-GlcNAc不那么受欢迎。在UDP存在或不存在的情况下，野生型B4Gal-T1与受体的结晶到目前为止都没有成功。这主要是由于开放构象中的apo-b4Gal-T1中没有受体结合位点。只有当UDP-Gal被结合时，该酶才以封闭的构象结晶，在那里存在受体位置。虽然UDP或受体分子可以诱导重要的构象变化，但到目前为止，此类络合物只有在LA存在的情况下才能结晶。由于LA与扩展的糖结合部位结合，因此在LA存在的情况下，b4Gal-T1不可能与寡糖受体结晶。