title: Structural Analysis of the Glycosylated Intact HIV-1 gp120-b12 Antibody Complex Using Hydroxyl Radical Protein Footprinting.
abstract: 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.
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Li
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Oliver C
authors.initials: OC
authors.lastname: Grant
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Keigo
authors.initials: K
authors.lastname: Ito
authors.affiliation: Department of Medicine, University of Massachusetts Medical School , Worcester, Massachusetts 01605, United States.
authors.forename: Aaron
authors.initials: A
authors.lastname: Wallace
authors.affiliation: Department of Medicine, University of Massachusetts Medical School , Worcester, Massachusetts 01605, United States.
authors.forename: Shixia
authors.initials: S
authors.lastname: Wang
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhao
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Lance
authors.initials: L
authors.lastname: Wells
authors.affiliation: Department of Medicine, University of Massachusetts Medical School , Worcester, Massachusetts 01605, United States.
authors.forename: Shan
authors.initials: S
authors.lastname: Lu
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Woods
authors.affiliation: Department of BioMolecular Sciences, University of Mississippi , University, Mississippi 38677, United States.
authors.forename: Joshua S
authors.identifier: 0000-0002-0115-0276
authors.initials: JS
authors.lastname: Sharp
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D057134:Antibodies, Neutralizing / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D015483:HIV Antibodies / Q000737:chemistry / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D017665:Hydroxyl Radical; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D000072417:Protein Domains; D023302:Protein Footprinting / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; C514793:gp120 protein, Human immunodeficiency virus 1; D017665:Hydroxyl Radical