Sructural study of immuno regulatory proteins by NMR spectroscopy

通过核磁共振波谱研究免疫调节蛋白的结构

• 批准号: 9357217
• 负责人: Ad Bax
• 金额: $ 22.57万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY 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/9357217
• 关键词: Affinity; Antigen-Presenting Cells; Binding; Blood Circulation; Cell surface; Complex; Detection; Engineering; Family; Family member; Genes; Homologous Gene; Immune system; Immunoglobulins; In Vitro; J segment gene; Length; Lymphoid Tissue; Major Histocompatibility Complex; Maps; Mediating; Murid herpesvirus 1; NMR Spectroscopy; Nucleotides; Pathway interactions; Peptides; Peripheral; Process; Protein NMR Spectroscopy; Proteins; Recombinants; Relaxation; Signal Transduction; Site; Staging; T-Cell Development; T-Cell Receptor; T-Lymphocyte; Techniques; Thymus Gland; Vertebral column; adaptive immunity; base; genetic regulatory protein; insight; polypeptide; research study

As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40, as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent immunoglobulin (Ig)-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full length and truncated forms of the MHC-I molecule H2-Ld by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-Ld, mini-H2-Ld, consisting of only the 12 platform domain. Mini-H2-Ld refolded in vitro with a high-affinity peptide to yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the 2-microglobulin (2m) interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and 2m-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the ps-ns dynamics of the free mini-H2-Ld MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway.

作为其通过宿主免疫系统逃避检测的策略的一部分，鼠巨细胞病毒（MCMV）编码了三种蛋白质，这些蛋白质调节了主要组织相容性复合物I类（MHC-I）分子的细胞表面表达：MHC-I同源物M152/GP40，以及M02-M16家族成员/GP4/M06/M06/M06/M06/MM06/MM06/MM06/MM04/MM04/MM04/MM04/MM06。先前对M04蛋白的研究表明，M02-M16家族的免疫消魔独有的免疫球蛋白（IG）类似折叠。在这里，我们通过多种技术来设计和表征重组M06，并以MHC-I分子H2-LD的全长和截短形式进行研究。此外，我们采用溶液NMR来绘制MHC-I上M06蛋白的相互作用足迹，利用截短的H2-LD，Mini-H2-LD，仅由12个平台域组成。 Mini-H2-LD用高亲和力的肽在体外重新折叠，以产生显示出色的NMR光谱特征的分子，从而允许完整的主链分配。这些基于NMR的研究表明，M06与位于肽结合平台下的离散位点紧密结合，该位置部分与MHC-I重链上的2-微球蛋白（2M）界面重叠，与体外结合实验一致，显示M06和2M辅助MHC-I-I-I之间的体外结合实验一致。此外，我们进行了NMR松弛实验，以表征游离Mini-H2-LD-LD MHC-I分子的PS-NS动力学，表明相互作用的位点被高度排序。这项研究提供了对M06与MHC-I相互作用的机理的见解，这表明在肽载荷途径的早期阶段对靶MHC-I分子进行了结构性操纵。