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: 8157137
• 负责人: PAUL T WINGFIELD
• 金额: $ 63.41万
• 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/8157137
• 关键词: Hepatitis B; Nucleocapsid Proteins; Structure

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. In an extension of this work, the stability of the capsids was probed 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 在大肠杆菌中表达，它在细菌细胞质中组装成二十面体衣壳，其中含有结合的宿主核酸。删除多碱基 C 端 34 个残基（鱼精蛋白结构域）也会产生装配能力蛋白。 C 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、图像分析和 X 射线晶体学确定。天然 HBeAg 也在第 149 位 C 端被截短，此外还包含源自前体蛋白 (pre-C) 部分加工的 10 个残基 N 端延伸。虽然 HBeAg 的功能和结构的详细知识尚不清楚，但它作为血清学标志物具有临床重要性。使用表面等离子共振 (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。鉴定了与衣壳的组装形式 (HBcAg) 和非组装形式 (HBeAg) 结合的单克隆抗体（鼠源），并描述了可用于临床诊断的新组合。先前通过冷冻电子显微镜对核衣壳-抗体免疫复合物进行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。这项工作已扩展到包括人类抗体，初步结果表明与衣壳表面的不同区域（表位）结合，我们之前使用模型鼠抗体识别了这些区域。这项工作与我们之前对衣壳免疫复合物和代表幼稚 B 细胞表面免疫球蛋白的抗体的描述一起，提供了抗体与与重要人类相关的病毒蛋白系统相互作用的最完整的结构图之一。疾病。 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，以与衣壳亚基高亲和力结合。抗体的结合阻止了衣壳（HBV 病毒的中心功能和结构成分）的组装，并且随着开发可能提供有用的抗 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特性受到限制。与 Albert Heck（乌得勒支大学）合作，使用大分子串联和离子淌度质谱法研究 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，并确定了 HBV 核衣壳复合物的精确分子化学计量。在这项工作的扩展中，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探测衣壳的稳定性。所使用的方法和途径对于纳米和生物技术中使用的大分子组装体的研究具有普遍适用性。