American Chemical Society
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Probing Heteromultivalent Protein–Glycosphingolipid Interactions using Native Mass Spectrometry and Nanodiscs

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journal contribution
posted on 2020-02-11, 22:29 authored by Ling Han, Pavel I. Kitov, Jianing Li, Elena N. Kitova, John S. Klassen
Interactions between glycosphingolipids (GSLs) on the surfaces of cells and glycan-binding proteins (GBPs) mediate a wide variety of essential and pathological processes. Despite the biological importance of these interactions, the GSL ligands of most GBPs remain to be identified and the mechanisms controlling recognition of GSLs are incompletely understood. Recently, it was suggested that, when present together with high affinity ligands, low affinity GSL ligands can contribute significantly to the binding of GBPs with multiple binding sites through a process called heteromultivalent binding. Here, with goal of directly establishing the existence of heteromultivalent GSL interactions and elucidating the mechanism underlying their formation, we investigated cholera toxin B subunit homopentamer (CTB5) binding to ganglioside mixtures in model membranes (nanodiscs) using native mass spectrometry (MS) and competitive ligand binding. Electrospray ionization (ESI)-MS analysis revealed that the presence of the high affinity ligand GM1 (at substoichiometric amounts relative to binding sites) in the nanodisc promotes GD1b binding to CTB5; no GD1b binding was detected in the absence of GM1. No direct ESI-MS evidence of CTB5 binding to the other five gangliosides tested, alone or present together with GM1 in the nanodiscs, was observed. Affinity measurements, carried out using the proxy ligand ESI-MS binding assay, confirmed that GD1b binding to CTB5 is dramatically enhanced (>1000-times higher affinity compared to the GD1b oligosaccharide affinity) when present with GM1. NDs containing GM1 and GM2, GD1a, or GT1b also exhibited enhanced CTB5 binding, however, the effect was smaller. The results of molecular dynamics simulations performed on ganglioside-containing nanodiscs suggest that the participation of low affinity ligands in heteromultivalent binding with GM1 may be regulated by the positions of the internal Gal-linked Neu5Ac residues of the gangliosides relative to the membrane surface.