Structure And Assembly Of The Hepatitis B Nucleocapsid Protein

乙型肝炎核衣壳蛋白的结构和组装

• 批准号: 7964902
• 负责人: PAUL T WINGFIELD
• 金额: $ 71.66万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 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/7964902
• 关键词: Address; Affinity; Antibodies; Antigen-Antibody Complex; Antigens; B-Lymphocytes; Binding; Biotechnology; C-terminal; Cancer Etiology; Capsid; Capsid Proteins; Childhood; Chronic Hepatitis B; Clinical; Code; Collaborations; Complex; Cryoelectron Microscopy; Cytoplasm; Detection; Development; Diagnostic; Drug Delivery Systems; Elasticity; Encapsulated; Epitopes; Fab Immunoglobulins; Genes; Hepatitis B; Hepatitis B Core Antigen; Hepatitis B Virus; Hepatitis B e Antigens; Heterogeneity; Human; Image Analysis; Intervention; Kinetics; Knowledge; Label; Libraries; Life Cycle Stages; Maps; Mass Spectrum Analysis; Measurement; Measures; Metabolic; Methods; Modeling; Molecular; Molecular Structure; Monoclonal Antibodies; Mus; N-terminal; Nanotechnology; Nucleic Acids; Nucleocapsid; Nucleocapsid Proteins; Oryctolagus cuniculus; Particulate; Play; Population; Positioning Attribute; Process; Property; Protamines; Protein Precursors; Proteins; RNA-Directed DNA Polymerase; Reagent; Role; Structure; Surface; Surface Immunoglobulins; Surface Plasmon Resonance; System; Time; Universities; Vaccines; Viral Proteins; Virus; Work; X-Ray Crystallography; anti-hepatitis B; clinical Diagnosis; hepatitis B virus P protein; human disease; ion mobility; molecular assembly/self assembly; prevent; protein S precursor; serological marker; stoichiometry; tool; virus core

HBcAg has been expressed in E.coli were it assembles in the bacterial cytoplasm into icosahedral capsids, which contain bound host nucleic acid. Deletion of the polybasic C-terminal 34 residues (protamine domain) also produces assembly competent protein. The capsids from C-terminal truncated protein (Cp149) do not contain nucleic acid and their structure has been previously determined by cryo-electron microscopy and image analysis and by X-ray crystallography. Native HBeAg is also C-terminally truncated at position 149 and in addition contains a 10 residue N-terminal extension derived from partial processing of precursor protein (pre-C). Although detailed knowledge of the function and structure of HBeAg are unknown it has clinical importance as a serological marker. Using surface plasmon resonance (Biacore) to measure antibody antigen interactions, a kinetic-affinity map of a panel of monoclonal antibodies (mAbs) against HBV nucleocapsid proteins was determined. Monoclonal antibodies (murine origin) binding to the assembled (HBcAg) and non-assembled forms of the capsids (HBeAg) were identified and new combinations useful for clinical diagnosis described. Previous structural determinations of nucleocapsid-antibody immune complexes by cryo-electron microscopy helped to more clearly explain the immunological distinction between the assembled HBcAg and unassembled HBeAg antigen. This work has been extended to included human antibodies and the preliminary results indicate binding to distinct regions on the capsid surface (epitopes) which we had previously identified using the model murine antibodies. This work, together our previous description of an immune complex of capsids and an antibody representative of the surface immunoglobulin from naive B-cells, provide one of the most complete structural pictures of the interaction of antibodies with a viral protein system related to an important human disease. Using a rabbit antibody library, monoclonal antibodies (mAbs) were selected then humanized to produce chimeric mAb fragment antigen binding portions (Fab). Fabs against the HBV capsid proteins were selected for high affinity binding to the capsid subunits. The binding of the antibodies prevent the assembly of the capsids (a central functional and structural component of the HBV virus) and may with development provide useful anti-HBV reagents. Direct determination of the biophysical properties of the HBV capsid protein is limited due to the size of the multiprotein megadalton complex. In collaboration with Albert Heck (Utrecht University) macromolecular tandem and ion mobility mass spectrometry was used to study the stability and conformational diversity of HBV capsids. Very precise mass measurements were made and the exact molecular stoichiometries of HBV nucleocapsid complexes determined for the first time. This work was extended to include measurements of stability and elasticity of the capsids which resulted in the detection of conformational heterogeneity in the capsid population. Also, the stability of the capsids was addressed by the direct determination of the slow exchange of constituent subunits using isotopically labeled protein. The methods and approaches used have general applicability to the study of large molecular assemblies used in nano- and biotechnology

HBcAg已在大肠杆菌中表达，其在细菌细胞质中组装成二十面体衣壳，其含有结合的宿主核酸。多碱基C-末端34个残基（鱼精蛋白结构域）的缺失也产生有组装能力的蛋白。来自C末端截短蛋白（Cp149）的衣壳不含核酸，其结构先前已通过冷冻电子显微镜和图像分析以及X射线晶体学确定。天然HBeAg也在位置149处被C-末端截短，并且另外含有源自前体蛋白（pre-C）的部分加工的10个残基的N-末端延伸。虽然HBeAg的功能和结构的详细知识是未知的，但它作为血清学标志物具有临床重要性。使用表面等离子体共振（Biacore）测量抗体抗原相互作用，确定一组针对HBV核衣壳蛋白的单克隆抗体（mAb）的动力学亲和力图。单克隆抗体（鼠源）结合组装（HBcAg）和非组装形式的衣壳（HBeAg）进行了鉴定，并描述了新的组合用于临床诊断。先前通过冷冻电子显微镜对核衣壳-抗体免疫复合物的结构测定有助于更清楚地解释组装的HBcAg和未组装的HBeAg抗原之间的免疫学区别。这项工作已经扩展到包括人抗体，并且初步结果表明与衣壳表面上的不同区域（表位）结合，我们先前使用模型鼠抗体鉴定了这些区域。这项工作，连同我们先前对衣壳的免疫复合物和代表来自幼稚B细胞的表面免疫球蛋白的抗体的描述，提供了抗体与重要人类疾病相关的病毒蛋白系统相互作用的最完整的结构图片之一。 使用兔抗体文库，选择单克隆抗体（mAb），然后人源化以产生嵌合mAb片段抗原结合部分（Fab）。选择针对HBV衣壳蛋白的Fab以高亲和力结合衣壳亚基。抗体的结合防止衣壳（HBV病毒的中心功能和结构组分）的组装，并且随着开发可以提供有用的抗HBV试剂。 由于多蛋白兆道尔顿复合物的大小，直接测定HBV衣壳蛋白的生物物理性质是有限的。与Albert Heck（乌得勒支大学）合作，使用大分子串联和离子迁移质谱法研究HBV衣壳的稳定性和构象多样性。进行了非常精确的质量测量，并首次确定了HBV核衣壳复合物的精确分子化学计量。这项工作被扩展到包括测量的稳定性和弹性的衣壳，导致在检测的衣壳人口的构象异质性。此外，通过使用同位素标记的蛋白质直接测定组成亚基的缓慢交换来解决衣壳的稳定性。所使用的方法和途径具有普遍适用性的研究大分子组装体中使用的纳米和生物技术