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.
Highlights
CD4 binding loop spanning residues 364−373 but involves many other residues.[10]
It was found that the removal of V1/V2 loops significantly weakens the binding of b12 to gp120.17 The removal of a single N-linked glycosylation site at the V3 loop increased the neutralization sensitivity of CD4 binding site (CD4bs) antibodies.[18]
To measure changes in high-resolution hydroxyl radical protein footprinting (HR-HRPF) reactivity, the stable modifications to the protein side chains are analyzed by liquid chromatography and mass spectrometry (LC−MS), relatively quantifying modified and unmodified peptides, and the MS signal of the oxidized version(s) of each peptide is compared to the MS signal of the unoxidized version of the same peptide to quantify oxidation at the peptide level
Summary
CD4 binding loop spanning residues 364−373 but involves many other residues.[10]. The truncated, deglycosylated, and mutationally stabilized gp[120] core is different from its mature counterpart in important ways, including a fully truncated V1/ V2 domain. Via measurement of the rate of reaction of each amino acid side chain of a protein under two different structural conditions (in this case, b12-bound vs free gp120), relative changes in the accessibility of each amino acid can be measured.[25] Previous work from our group has shown that, using ETD-based methods for quantifying multiple adjacent sites of isomeric oxidation products, we can accurately quantify changes in the hydroxyl radical footprint down to single-amino acid spatial resolution,[30,31] providing structural information with higher spatial resolution and more accuracy This method offers an important alternative method for characterizing protein−protein and protein−ligand interactions in cases in which it is not possible to determine high-resolution three-dimensional structures of complexes because of the heterogeneity, flexibility, and/or size of the target(s). This provided a structure-based rationalization of the experimentally determined protection sites as well as detailed insights into the regions of JR-FL affected by complexation with b12
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