STRUCTURAL STUDIES OF CLASS II MHC PROTEINS

II 类 MHC 蛋白质的结构研究

• 批准号: 2330447
• 负责人: Lawrence J. Stern
• 金额: $ 14.06万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2330447
• 关键词: MHC class II antigen; Sf9 cell line; X ray crystallography; antigen presentation; autoantigens; biophysics; chemical binding; computer simulation; conformation; crystallization; electrospray ionization mass spectrometry; hydrogen bond; intermolecular interaction; microorganism hemagglutinin; peptides; protein folding; protein purification; protein structure function; structural biology; synthetic peptide

The proposed research is an investigation into molecular aspects of antigen selection and antigen presentation by class II major histocompatibility (MHC) proteins. The human class II MHC proteins HLA- DR1, DR3, and DQ8 will be expressed as empty molecules in insect cells and loaded in vitro with antigenic peptides. Three-dimensional structures will be determined by X-ray crystallography for two or more peptide complexes of DR1 and for peptide complexes of DR3 and DQ8. Currently the three- dimensional structure of only one class II MHC-peptide complex is known. The proposed structural determinations for peptide complexes of DR1 and DR3 will indicate the generality of the peptide binding mechanism observed in the published DR1-HA peptide structure and the source of the observed differences in peptide binding preferences for DR1 and DR3. The proposed structural determinations for peptide complexes of DR3 and DQ8 will identify any unusual features of these proteins that may be responsible for their disease association. In addition, these structures will provide a framework for interpretation of the large amount of data concerning antigen selection and presentation by other class II MHC proteins and will improve our ability to predict the peptide binding preferences of MHC proteins and the potential antigenicity of peptide and protein therapeutics. Detailed information about the peptide binding site will aid efforts to design structure-based antagonists and agonists of the MHC- antigen-T-cell receptor interactions. Class II MHC proteins will be expressed in E. coli and refolded in vitro, as an alternative to the insect cell expression system. Development of the E. coli expression system will provide a rich source of class II MHC proteins and class II- peptide complexes for crystallographic and other biophysical investigations of the peptide binding mechanism. Preliminary expression studies have provided DR1, DR3, and DQ8 proteins. Preliminary crystallographic studies have provided crystals of HLA-DR1 in complex with an endogenous peptide derived from HLA-A2, and initial phase information for determination of the structure of the DR1-A2 peptide complex. These crystals grow in a new crystal form that diffracts to higher resolution than previously reported class II MHC crystals. Preliminary refolding studies have produced active DR1 in approximately 30% yield after expression in E. coli and refolding in vitro.

拟议的研究是对分子方面的调查 II类专业的抗原选择和抗原呈递 组织相容性 (MHC) 蛋白。人类 II 类 MHC 蛋白 HLA- DR1、DR3和DQ8将在昆虫细胞中表达为空分子， 体外负载抗原肽。三维结构将 通过 X 射线晶体学测定两个或多个肽复合物 DR1 以及 DR3 和 DQ8 的肽复合物。目前，三 仅一种 II 类 MHC 肽复合物的空间结构是已知的。 DR1 和 DR1 肽复合物的拟议结构测定 DR3 将表明观察到的肽结合机制的普遍性 已发表的 DR1-HA 肽结构和观察到的来源 DR1 和 DR3 肽结合偏好的差异。拟议的 DR3 和 DQ8 肽复合物的结构测定将 识别这些蛋白质的任何异常特征可能是造成这种情况的原因 他们的疾病关联。此外，这些结构将提供 一个解释大量数据的框架 由其他 II 类 MHC 蛋白进行抗原选择和呈递，并将 提高我们预测 MHC 肽结合偏好的能力 蛋白质以及肽和蛋白质的潜在抗原性 疗法。有关肽结合位点的详细信息将有助于 努力设计基于结构的 MHC 拮抗剂和激动剂 抗原-T 细胞受体相互作用。 II 类 MHC 蛋白将是 在大肠杆菌中表达并在体外重折叠，作为 昆虫细胞表达系统。大肠杆菌表达的开发 系统将提供丰富的 II 类 MHC 蛋白和 II 类- 用于晶体学和其他生物物理的肽复合物 肽结合机制的研究。初步表达 研究提供了 DR1、DR3 和 DQ8 蛋白。初步的 晶体学研究提供了 HLA-DR1 的晶体 来自 HLA-A2 的内源性肽和初始相信息 用于确定 DR1-A2 肽复合物的结构。这些 晶体以新的晶体形式生长，衍射分辨率更高 比之前报道的 II 类 MHC 晶体。初步重折叠 研究表明，活性 DR1 的产率约为 30% 在大肠杆菌中表达并在体外重折叠。