Crystallographic studies of macromolecular structures

大分子结构的晶体学研究

• 批准号: 8149115
• 负责人: Lars Pedersen
• 金额: $ 85.64万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 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/8149115
• 关键词: Molecular Structure

Independently our lab is focused on the development of specific sulfotransferases to be used in enzymatic production of therapeutic heparan sulfate. Heparan sulfates (HS) are linear sulfated polysaccharides present on the cell surface and in the extra cellular matrix that play important roles in blood coagulation, inflammation response, cell differentiation and assist in bacterial and viral infection. The specific sulfation pattern of HS determines its functional selectivity. Different sulfotransferases are required for sulfation of specific hydroxyl groups or amines along the polysaccharide. Heparin is a highly sulfated form of HS. Theraputic heparin is a 3 billion dollar a year industry as an anticoagulant. In addition low molecular weight heparin/heparan sulfate mimics show promise as potential anti-cancer/anti-metastasis drugs possibly due to their role in growth factor regulation and as heparanase inhibitors. Currently therapeutic heparin is purified from mast cells of mammalian sources. This can lead to contamination problems as well as and perhaps more importantly heterogeneity problems. One of the major side-effects of administration of heparin is thrombocytopenia due to interaction of the heparin with platelet factor 4 (PF4). Chemical synthesis of homogeneous polysaccharides larger than a hexasaccharide is extremely difficult and currently too challenging for mass production. In collaboration with the Liu lab in the School of Pharmacy at UNC we use structural guided mutagenesis to understand the specificity of the heparan sulfate sulfotransferases. If we are able to redesign these enzymes we can tailor the production of task specific heparan sulfates which are more homogenous and have fewer side effects. Another focus of the Structure Function Group is to use X-ray crystallography to support research interest of principal investigators within the intramural community. One such collaboration is with Geoffrey Mueller in the London group studying crystal structures of dust mite allergens. This year we published the crystal structures of two dust mite allergens, Der p 7 and Der p 5. Skin tests have shown reactivity of Der p 7 in 53% of allergic patients yet very little is known about this protein. The crystal structure has revealed a BPI domain-like fold suggesting a possible role as a lipid transporter for lipids such as LPS. The crystal structure of Der p 5 is a three helical bundle and resolved a dispute in the literature with regard to the ordering of the helices for Group 5 allergens. The structure suggests that Der p 5 also has the potential to bind hydrophobic ligands and suggests a potential to form higher ordered oligomers. In theory, structural information on these allergens could allow for the design of recombinant hypoallergenic proteins for immunotherapy. In addition, we have extensive collaborations with the Wilson and Kunkel laboratories studying crystal structures of polymerases involved in DNA repair process. In collaboration we continue to help determine structures of human X-family polymerases beta and lambda to better understand the role of these enzymes in base excision and non-homologous end-joining repair processes. This past year we also published on a technique that we developed which appears to improve the likelihood of obtaining diffraction quality crystals from recalcitrant proteins. Combining the techniques of MBP-fusion/fix-arm crystallization with surface entropy reduction, we have developed a suite of vectors with different mutations on the surface of the MBP that allows one to screen for a specific target using multiple surfaces that can form potential crystal lattice contacts. This technique has allowed us to solve the structure of five proteins in the lab that we were unable to obtain structures of prior to applying this technique.

我们的实验室独立致力于开发用于酶法生产治疗性硫酸乙酰肝素的特定磺基转移酶。 硫酸乙酰肝素 (HS) 是存在于细胞表面和细胞外基质中的线性硫酸化多糖，在凝血、炎症反应、细胞分化以及协助细菌和病毒感染中发挥重要作用。 HS 的特定硫酸化模式决定了其功能选择性。 多糖上的特定羟基或胺的硫酸化需要不同的磺基转移酶。肝素是 HS 的高度硫酸化形式。 治疗性肝素作为抗凝剂是一个年产值 30 亿美元的产业。 此外，低分子量肝素/硫酸乙酰肝素模拟物有望成为潜在的抗癌/抗转移药物，这可能是由于它们在生长因子调节中的作用以及作为乙酰肝素酶抑制剂的作用。 目前，治疗性肝素是从哺乳动物来源的肥大细胞中纯化的。 这可能导致污染问题以及可能更重要的异质性问题。 肝素给药的主要副作用之一是由于肝素与血小板因子 4 (PF4) 相互作用而导致的血小板减少。 化学合成大于六糖的均质多糖极其困难，目前对于大规模生产来说太具有挑战性。 我们与北卡罗来纳大学药学院刘实验室合作，使用结构引导诱变来了解硫酸乙酰肝素磺基转移酶的特异性。 如果我们能够重新设计这些酶，我们就可以定制任务特异性硫酸乙酰肝素的生产，这种硫酸乙酰肝素更均匀且副作用更少。 结构功能小组的另一个重点是使用 X 射线晶体学来支持校内社区内主要研究人员的研究兴趣。 其中一项合作是与伦敦小组的杰弗里·米勒（Geoffrey Mueller）合作，研究尘螨过敏原的晶体结构。 今年，我们发表了两种尘螨过敏原 Der p 7 和 Der p 5 的晶体结构。皮肤测试显示 53% 的过敏患者具有 Der p 7 的反应性，但人们对这种蛋白质知之甚少。 该晶体结构揭示了类似 BPI 结构域的折叠，表明其可能作为 LPS 等脂质的脂质转运蛋白。 Der p 5 的晶体结构是三螺旋束，解决了文献中关于第 5 组过敏原的螺旋顺序的争议。 该结构表明 Der p 5 还具有结合疏水性配体的潜力，并表明具有形成更高有序寡聚物的潜力。 理论上，这些过敏原的结构信息可以允许设计用于免疫治疗的重组低过敏性蛋白。 此外，我们与 Wilson 和 Kunkel 实验室进行了广泛的合作，研究参与 DNA 修复过程的聚合酶的晶体结构。 通过合作，我们继续帮助确定人类 X 家族聚合酶 β 和 lambda 的结构，以更好地了解这些酶在碱基切除和非同源末端连接修复过程中的作用。 去年，我们还发表了我们开发的一项技术，该技术似乎提高了从顽固蛋白质中获得衍射质量晶体的可能性。 将MBP融合/固定臂结晶技术与表面熵减少相结合，我们开发了一套在MBP表面具有不同突变的载体，允许使用可以形成潜在晶体的多个表面来筛选特定目标晶格接触。 这项技术使我们能够在实验室中解析五种蛋白质的结构，而在应用该技术之前我们无法获得这些蛋白质的结构。