Detection and Quantitative Analysis of Two Independent Binding Modes of a Small Ligand Responsible for DC-SIGN Clustering

Abstract

DC-SIGN (dendritic cell-specific ICAM-3 grabbing non-integrin) is a C-type lectin receptor (CLR) present, mainly in dendritic cells (DCs), as one of the major pattern recognition receptors (PRRs). This receptor has a relevant role in viral infection processes. Recent approaches aiming to block DC-SIGN have been presented as attractive anti-HIV strategies. DC-SIGN binds mannose or fucose-containing carbohydrates from viral proteins such as the HIV envelope glycoprotein gp120. We have previously demonstrated that multivalent dendrons bearing multiple copies of glycomimetic ligands were able to inhibit DC-SIGN-dependent HIV infection in cervical explant models. Optimization of glycomimetic ligands requires detailed characterization and analysis of their binding modes because they notably influence binding affinities. In a previous study we characterized the binding mode of DC-SIGN with ligand 1, which shows a single binding mode as demonstrated by NMR and X-ray crystallography. In this work we report the binding studies of DC-SIGN with pseudotrisaccharide 2, which has a larger affinity. Their binding was analysed by TR-NOESY and STD NMR experiments, combined with the CORCEMA-ST protocol and molecular modelling. These studies demonstrate that in solution the complex cannot be explained by a single binding mode. We describe the ensemble of ligand bound modes that best fit the experimental data and explain the higher inhibition values found for ligand 2.