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.

独立地，我们的实验室专注于开发特定的磺胺转移酶，用于用于酶促硫酸乙酰肝素的酶产生。 乙酰硫酸盐（HS）是细胞表面和额外细胞基质中存在的线性硫酸盐多糖，在血液凝结，炎症反应，细胞分化以及有助于细菌和病毒感染中起着重要作用。 HS的特定硫酸化模式决定了其功能选择性。 沿多糖的特定羟基或胺的硫酸化需要不同的硫代转移酶。肝素是高度硫化的HS。 治疗肝素是每年30亿美元的抗凝剂行业。 另外，低分子量肝素/硫酸肝素模拟物显示出可能是由于它们在生长因子调节中的作用和作为肝素酶抑制剂所致的潜在抗癌/抗震源药物。 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更重要的是异质性问题。 肝素给药的主要副作用之一是血小板减少症，这是由于肝素与血小板因子4的相互作用（PF4）。 大于六糖的均质多糖的化学合成非常困难，目前对于群众生产而言太具有挑战性。 在UNC的药学院与LIU实验室合作，我们使用结构引导的诱变来了解硫酸乙酰肝素硫酸盐硫酸盐转移酶的特异性。 如果我们能够重新设计这些酶，我们可以定制特定于任务的乙酰基硫酸盐的产生，乙酰肝素硫酸盐更均匀，副作用较少。 结构功能组的另一个重点是使用X射线晶体学来支持壁内社区中主要研究人员的研究兴趣。 这样的合作是与杰弗里·穆勒（Geoffrey Mueller）在伦敦小组中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的晶体结构，Der p 7和der p 5。皮肤测试显示了53％的过敏患者的反应性，但对此蛋白质知之甚少。 晶体结构揭示了BPI结构域的折叠，这表明作为脂质（例如LPS）的脂质转运蛋白可能起作用。 Der P 5的晶体结构是三个螺旋束，并在文献中就5组过敏原的螺旋序列进行了争议。 该结构表明，der p 5也具有结合疏水配体的潜力，并提出了形成较高有序低聚物的潜力。 从理论上讲，这些过敏原的结构信息可以允许设计重组低过敏蛋白进行免疫疗法。 此外，我们与Wilson和Kunkel实验室进行了广泛的合作，研究了参与DNA修复过程的聚合酶的晶体结构。 在合作中，我们继续帮助确定人类X-家庭聚合酶Beta和Lambda的结构，以更好地了解这些酶在碱基切除和非同源最终结合修复过程中的作用。 在过去的一年中，我们还发表了我们开发的一种技术，该技术似乎改善了从顽固蛋白中获得衍射质量晶体的可能性。 将MBP融合/固定臂结晶的技术与表面熵的还原相结合，我们开发了一套在MBP表面上具有不同突变的向量，使人们可以使用可以形成潜在的晶体晶格接触的多个表面筛选特定靶标。 这项技术使我们能够解决实验室中五种蛋白质的结构，在应用此技术之前，我们无法获得该结构。