Li Xiaoyan, Grant Oliver C, Ito Keigo, Wallace Aaron, Wang Shixia, Zhao Peng, Wells Lance, Lu Shan, Woods Robert J, Sharp Joshua S
Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
Department of Medicine, University of Massachusetts Medical School , Worcester, Massachusetts 01605, United States.
Biochemistry. 2017 Feb 21;56(7):957-970. doi: 10.1021/acs.biochem.6b00888. Epub 2017 Feb 6.
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.
糖蛋白gp120是人类免疫缺陷病毒1型的表面抗原和毒力因子。如果能够理解gp120与广泛中和抗体（bNAbs）之间的相互作用，那么对来自多种HIV分离株的gp120产生反应的广泛中和抗体有望用于开发具有广泛效力的免疫原以用于疫苗接种。从结构角度来看，gp120是一个特别复杂的系统，因其尺寸较大、存在多个柔性区域以及大量糖基化修饰，所有这些在gp120与bNAbs的相互作用中都很重要。在此，通过蛋白质的快速光化学氧化，利用高分辨率羟基自由基蛋白质足迹法（HR-HRPF）探究全长糖基化gp120与bNAb b12的相互作用。HR-HRPF能够测量多个氨基酸的平均溶剂可及表面积的变化，而无需采取可能改变蛋白质构象的措施，如诱变。gp120-b12复合物的HR-HRPF结合计算建模显示V1/V2结构域存在一种新的广泛相互作用，可能是与b12的轻链相互作用。我们的数据还揭示了N330聚糖与b12轻链相互作用导致C3结构域出现HR-HRPF保护现象。除了提供关于全长糖基化gp120与b12相互作用的信息外，这项工作还为全长糖基化gp120与其他bNAbs的结构研究提供了模板，以便更好地表征驱动bNAb广泛特异性的相互作用。
