Structural Insights Into Man₉ Recognition by the HIV Antibody 2G12 Revealed by Paramagnetic NMR

Viral proteins contain "glycoepitopes" that can be targeted by antibodies. A prominent example is HIV gp120 glycoprotein, which contains a dense cluster of oligomannoside glycans recognized by the broadly neutralizing 2G12 antibody. 3D structures of 2G12 complexed with oligomannosides of diverse sizes have been determined using X-ray crystallography and cryo-electron microscopy, and different glycan binding epitopes have been proposed, with either the D1 arm or the D3 arm of the glycans establishing the main interactions with the antibody. In this work, we have synthesized a Man₉ derivative bearing a lanthanide binding tag to perform detailed characterization of the glycan binding epitope in solution using NMR under paramagnetic conditions. This approach allows studying the Man₉ structure, overcoming the signal overlapping problems found in standard NMR experiments. STD NMR analysis revealed a clear preference for the D1 arm in 2G12 antibody recognition, while the D2 and D3 branches also contributed to binding, albeit with weaker STD effects. This relevant information is fundamental for the design of immunogens to elicit 2G12-like antibodies as vaccine components, since recent studies have found that these "glycoepitopes" are also present in other viruses such as SARS-CoV-2 and certain H3N2 influenza strains.