Structural Analysis of the Glycosylated Intact HIV-1 gp120-b12 Antibody Complex Using Hydroxyl Radical Protein Footprinting.
Structural Analysis of the Glycosylated Intact HIV-1 gp120-b12 Antibody Complex Using Hydroxyl Radical Protein Footprinting.
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DOI：
10.1021/acs.biochem.6b00888
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发表时间：
2017-02-21
期刊：
影响因子：
2.9
通讯作者：
Sharp JS
中科院分区：
文献类型：
--
作者：
Li X;Grant OC;Ito K;Wallace A;Wang S;Zhao P;Wells L;Lu S;Woods RJ;Sharp JS
糖蛋白gp120是人类免疫缺陷病毒1的一种表面抗原和毒力因子。广谱中和抗体(BNAbs)与来自各种HIV分离株的gp120发生反应，如果能够理解gp120和bNAbs之间的相互作用，就有希望开发出广泛有效的免疫原用于疫苗接种。从结构的角度来看，gp120是一个特别困难的系统，因为它的大小，存在多个柔性区域，以及大量的糖基化，所有这些在gp120-bNAb相互作用中都是重要的。在这里，全长，糖基化的gp120与bNAb B12的相互作用被用高分辨率羟基自由基蛋白足迹(HR-HRPF)通过蛋白质的快速光化学氧化来探索。HR-HRPF允许测量多种氨基酸的平均溶剂可及表面积的变化，而不需要采取可能改变蛋白质构象的措施，例如突变。Gp120-B12复合体的HR-HRPF与计算模型相结合，显示了V1/V2结构域的广泛相互作用，可能与B12的轻链有关。我们的数据还表明，由于N330糖链与B12轻链的相互作用，C3区域的HR-HRPF具有保护作用。除了提供全长糖基化gp120与B12相互作用的信息外，本工作还为全长糖基化gp120与其他bNAb的结构询问提供了模板，以更好地表征驱动bNAb广泛特异性的相互作用。
Glycoprotein gp120 is a surface antigen and virulence factor of human immunodeficiency virus 1. Broadly neutralizing antibodies (bNAbs) that react to gp120 from a variety of HIV isolates offer hope for the development of broadly effective immunogens for vaccination purposes, if the interactions between gp120 and bNAbs can be understood. From a structural perspective, gp120 is a particularly difficult system because of its size, the presence of multiple flexible regions, and the large amount of glycosylation, all of which are important in gp120–bNAb interactions. Here, the interaction of full-length, glycosylated gp120 with bNAb b12 is probed using high-resolution hydroxyl radical protein footprinting (HR-HRPF) by fast photochemical oxidation of proteins. HR-HRPF allows for the measurement of changes in the average solvent accessible surface area of multiple amino acids without the need for measures that might alter the protein conformation, such as mutagenesis. HR-HRPF of the gp120–b12 complex coupled with computational modeling shows a novel extensive interaction of the V1/V2 domain, probably with the light chain of b12. Our data also reveal HR-HRPF protection in the C3 domain caused by interaction of the N330 glycan with the b12 light chain. In addition to providing information about the interactions of full-length, glycosylated gp120 with b12, this work serves as a template for the structural interrogation of full-length glycosylated gp120 with other bNAbs to better characterize the interactions that drive the broad specificity of the bNAb.