Crystallographic studies of macromolecular structures

大分子结构的晶体学研究

基本信息

批准号：
10249875
负责人：
Lars Pedersen
金额：
$ 145.31万
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
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/10249875
关键词：
2019-nCoV ATP phosphohydrolase Active Sites Address Affect Amino Acids Anabolism Antibodies Antigens Behavior Binding COVID-19 Catalysis Cell Death Cells Chondroitin Sulfate A Chondroitin Sulfates Chromosome Pairing Chromosomes Coagulation Process Collaborations Communities Complex Cryoelectron Microscopy Crystallization Crystallography DNA DNA Damage DNA Double Strand Break DNA Repair DNA biosynthesis DNA polymerase mu Disease Double Strand Break Repair Effectiveness Enzymes Exposure to Facioscapulohumeral Muscular Dystrophy Family Gene Rearrangement Gene Silencing Genomic Imprinting Goals Health Heparan Sulfate Biosynthesis Human Hydrogen Bonding Hydroxyl Radical Immunoglobulin Class Switching Immunoglobulin Switch Recombination Inflammation Insect Proteins Ionizing radiation Lead Length Macromolecular Complexes Maintenance Malignant Neoplasms Mediating Missense Mutation Molecular Conformation Molecular Structure Mutation National Institute of Environmental Health Sciences Nonhomologous DNA End Joining Nuclear Receptors Nucleotides Oligosaccharides Pattern Polymerase Process Production Protein Conformation Protein Isoforms Proteins Reactive Oxygen Species Reporting Research Research Support Resolution Roentgen Rays Role SARS coronavirus Severe Acute Respiratory Syndrome Site Specificity Structural Protein Structure Sulfate Techniques Therapeutic Time Virus Diseases Work X Inactivation biological systems dimer enzyme biosynthesis experimental study flexibility folic acid metabolism human coronavirus improved insight interest mu opioid receptors mutant novel coronavirus novel therapeutics protein complex protein function repaired scaffold sulfotransferase tool

项目摘要

Our lab works on a variety of different biological systems including heparan/chondroitin sulfate biosynthesis, DNA replication/repair, nuclear receptors, folate metabolism, and antibody/antigen interactions. Listed below are a few of our main projects form last year: 1) Work on glycosylaminoglycan (GAG) biosynthesis (in collaboration with Dr. Jian Liu at UNC) was centered around GAG biosynthesis enzymes that are utilized for the production of potential therapeutics dealing with coagulation, inflammation, and viral infection. By understanding how these enzymes interact with their substrates, we hope to modify them to improve their effectiveness in generating novel therapeutics with specific lengths, sequences, and sulfation patterns. CHST15 is a sulfotransferase that transfers a sulfate to the C-6 hydroxyl of GalNAc4S of chondroitin sulfate A to produce chondroitin sulfate E (CS-E). We have been able to obtain soluble protein from insect cells and partially deglycosylate it, finally giving us good material for ongoing crystallization studies. In addition, we have focused on structures of sulfotransferases involved in heparan sulfate biosynthesis. To this end, we have solved a co-crystal structure of 3-O-sulfotransferase (3-OST) isoform 5 in the presence of an oligosaccharide to mid-resolution. This structure allows for comparisons of substrate binding to isoforms 1 and 3 for which we have already determined structures, to help us better understand the specificity differences between these enzymes. Finally, our structures have directed research demonstrating that a triple mutant of 6-O-sulfotransferase, generated in our lab, only sulfates the non-reducing end of HS chains. This mutant provides a critical tool for the synthesis of homogenous HS oligosaccharides with specific 6-O-sulfation. 2) Double-strand breaks in DNA can result from exposure to ionizing radiation, reactive oxygen species, and other DNA damaging agents. Such damage can lead to cell death or cancer. Non-homologous End Joining (NHEJ) is a favored form of double-strand break (DSB) repair and is critical in non-replicating cells. NHEJ is also utilized in immunoglobin gene rearrangement and class-switch recombination. In support of Dr. Thomas Kunkels Replication Fidelity Group at NIEHS, our work this year was focused on understanding how X family polymerase Mu (pol mu) engages and repairs DSBs. Andrea Kaminski in our group was able to obtain pre-, intermediate and post-catalytic structures of pol mu bound to a DNA DSB. These represent the first structures from either pol mu or lambda bound to a DSB. Synapsis is mediated solely by Pol, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Pol may address complementary DSB substrates during NHEJ in a manner nearly indistinguishable from single-strand breaks, with subtle conformational changes in N457 and H459 near the break. This supports the notion that the rigid Pol mu scaffold provides for great flexibility in the types of substrates it can support for catalysis to repair damaged DNA. In addition, we worked in support of Dr. Samuel Wilsons group at NIEHS to critically refine and validate an array of time-lapse crystallography structures of Pol lambda (also an X-family polymerase) to investigate the process of correct nucleotide incorporation compared to incorporation of a mispair. These results demonstrate that misincorporation proceeds through a mostly open state of the enzyme that more closely resembles the binary rather than the ternary complex. 3) SMCHD1 (structural maintenance of chromosome flexible hinge domain containing 1) is a 2005 amino acid protein involved in gene silencing, with reported roles in X inactivation, genomic imprinting, and non-homologous end joining (NHEJ). Missense mutations in SMCHD1 are associated with arhinia, and Facioscapulohumeral Muscular Dystrophy Type 2 (FSHD2). In supporting the research by Dr. Natalie Shaw at NIEHS, we are trying to understand how these mutations lead to disease. Having previously solved the crystal structure of the ATPase module of SMCHD1, this year we focused on solving crystal structures of mutants and started CryoEM experiments to address how various mutations may affect the conformational landscape of SMCHD1 function. The mutations we investigated have surprisingly provided no changes in the global conformation of the protein in the dimeric state. This project involves research on human coronavirus, novel coronavirus, COVID-19, Severe Acute Respiratory Syndrome coronavirus disease, SARS coronavirus, SARS-coronavirus-2, SARS-cov-2, SARS-cov2, SARS-related coronavirus 2, Severe acute respiratory syndrome coronavirus 2, SARS-Associated Coronavirus, SARS-cov, or SARS-Related Coronavirus.

我们的实验室致力于各种不同的生物系统，包括乙酰肝素/硫酸软骨素生物合成、DNA 复制/修复、核受体、叶酸代谢和抗体/抗原相互作用。以下列出了我们去年的一些主要项目： 1) 糖胺聚糖 (GAG) 生物合成方面的工作（与北卡罗来纳大学的刘健博士合作）以 GAG 生物合成酶为中心，该酶用于生产治疗凝血、炎症和病毒感染的潜在疗法。通过了解这些酶如何与其底物相互作用，我们希望对其进行修饰，以提高其生成具有特定长度、序列和硫酸化模式的新型疗法的有效性。 CHST15 是一种磺基转移酶，可将硫酸盐转移至硫酸软骨素 A 的 GalNAc4S 的 C-6 羟基上，产生硫酸软骨素 E (CS-E)。我们已经能够从昆虫细胞中获得可溶性蛋白质，并将其部分去糖基化，最终为我们正在进行的结晶研究提供了良好的材料。此外，我们还关注参与硫酸乙酰肝素生物合成的磺基转移酶的结构。为此，我们在寡糖存在下以中等分辨率解析了 3-O-磺基转移酶 (3-OST) 亚型 5 的共晶结构。该结构允许比较与我们已经确定结构的亚型 1 和 3 结合的底物，以帮助我们更好地了解这些酶之间的特异性差异。最后，我们的结构指导研究表明，我们实验室生成的 6-O-磺基转移酶三重突变体仅硫酸化 HS 链的非还原端。该突变体为合成具有特定 6-O-硫酸化的均质 HS 寡糖提供了关键工具。 2) DNA 双链断裂可能是由于暴露于电离辐射、活性氧和其他 DNA 损伤剂造成的。这种损害可能导致细胞死亡或癌症。非同源末端连接 (NHEJ) 是一种受欢迎的双链断裂 (DSB) 修复形式，对于非复制细胞至关重要。 NHEJ 还用于免疫球蛋白基因重排和类别转换重组。为了支持 NIEHS 的 Thomas Kunkels 复制保真小组博士，我们今年的工作重点是了解 X 家族聚合酶 Mu (pol mu) 如何参与和修复 DSB。我们小组的 Andrea Kaminski 能够获得与 DNA DSB 结合的 pol mu 的催化前、中间和催化后结构。这些代表了与 DSB 结合的 pol mu 或 lambda 的第一个结构。突触仅由 Pol 介导，由断裂位点的单核苷酸同源性促进，其中不连续模板链的两端通过氢键网络稳定。四元 Pol 复合物中的活性位点准备好进行催化，并且核苷酸掺入在晶体中进行。这些结构表明，Pol 可以在 NHEJ 过程中以与单链断裂几乎无法区分的方式处理互补 DSB 底物，在断裂附近 N457 和 H459 中的构象发生细微变化。这支持了这样的观点，即刚性 Pol mu 支架在底物类型方面提供了极大的灵活性，它可以支持催化修复受损 DNA。此外，我们在 NIEHS 的 Samuel Wilsons 博士小组的支持下，严格精炼和验证了 Pol lambda（也是 X 家族聚合酶）的一系列延时晶体学结构，以研究与掺入相比正确的核苷酸掺入过程的错配。这些结果表明，错误掺入是通过酶的大部分开放状态进行的，该状态更类似于二元复合物而不是三元复合物。 3) SMCHD1（含有 1 的染色体柔性铰链结构域的结构维持）是一种 2005 年氨基酸蛋白，参与基因沉默，据报道在 X 失活、基因组印记和非同源末端连接 (NHEJ) 中发挥作用。 SMCHD1 的错义突变与 Arhinia 和 2 型面肩肱型肌营养不良症 (FSHD2) 相关。为了支持 NIEHS 的 Natalie Shaw 博士的研究，我们试图了解这些突变如何导致疾病。之前已经解决了 SMCHD1 ATPase 模块的晶体结构，今年我们专注于解决突变体的晶体结构，并开始 CryoEM 实验，以解决各种突变如何影响 SMCHD1 功能的构象景观。令人惊讶的是，我们研究的突变没有使二聚体状态的蛋白质的整体构象发生变化。该项目涉及人类冠状病毒、新型冠状病毒、COVID-19、严重急性呼吸系统综合症冠状病毒病、SARS冠状病毒、SARS-coronavirus-2、SARS-cov-2、SARS-cov2、SARS相关冠状病毒2、严重急性呼吸道疾病的研究综合征冠状病毒 2、SARS 相关冠状病毒、SARS-cov 或 SARS 相关冠状病毒。