Structural Characterization of the DC-SIGN–Lewis^X Complex

Abstract

Dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is a C-type lectin highly expressed on the surface of antigen-presenting dendritic cells. DC-SIGN mediates interactions among dendritic cells, pathogens, and a variety of epithelia, myeloid cells, and endothelia by binding to high mannose residues on pathogenic invaders or fucosylated residues on the membranes of other immune cells. Although these interactions are normally beneficial, they can also contribute to disease. The structural characterization of binding geometries is therefore of interest as a basis for the construction of mimetics that can mediate the effects of abnormal immune response. Here, we report the structural characteristics of the interaction of the DC-SIGN carbohydrate recognition domain (CRD) with a common fucosylated entity, the LewisX trisaccharide (LeX), using NMR methods. Titration of the monomeric DC-SIGN CRD with LeX monitored by 2D NMR revealed significant perturbations of DC-SIGN cross-peak positions in 1H–15N heteronuclear single quantum coherence (HSQC) spectra and identified residues near the binding site. Additionally, saturation transfer difference (STD) and transferred nuclear Overhauser effect (trNOE) NMR experiments, using a tetrameric form of DC-SIGN, identified binding epitopes and bound conformations of the LeX ligand. The restraints derived from these multiple experiments were used to generate models for the binding of LeX to the DC-SIGN CRD. Ranking of the models based on the fit of model-based simulations of the trNOE data and STD buildup curves suggested conformations distinct from those seen in previous crystal structures. The new conformations offer insight into how differences between binding of LewisX and mannose-terminated saccharides may be propagated.

Identification of Multiple Druggable Secondary Sites by Fragment Screening against DC‐SIGN

“…Abstract: DC-SIGN is ac ell-surface receptor for several pathogenic threats,s uch as HIV,E bola virus,o rM ycobacterium tuberculosis.M ultiple attempts to develop inhibitors of the underlying carbohydrate-protein interactions have been undertaken in the past fifteen years.S till, drug-like DC-SIGN ligands are sparse,w hich is most likely due to its hydrophilic, solvent-exposed carbohydrate-binding site.H erein, we report on ap arallel fragment screening against DC-SIGN applying SPR and ar eporter displacement assay, which complements previous screenings using 19 FNMR spectroscopyand chemical fragment microarrays.H it validation by SPR and 1 H- 15 N HSQC NMR spectroscopyrevealed that although no fragment bound in the primary carbohydrate site,five secondary sites are available to harbor drug-like molecules.B uilding on key interactions of the reported fragment hits,these pockets will be targeted in future approaches to accelerate the development of DC-SIGN inhibitors.…”

“…[11] It is noteworthy that only four compounds were active in the RDAwith IC 50 values above 1mm.A st his assay directly probes for competitors rather than binders,i ti mplies low druggability for the carbohydrate-binding site. To expand our insight into the presence of secondary sites, we studied 15 N-labeled DC-SIGN CRD by 1 H-15 N HSQC NMR spectroscopy (Figure 2a) [15] and applied computational pocket predictions.T he titration of mannose, am illimolar ligand, led to chemical shift perturbations (CSPs) and reduced resonance intensities for residues close to the primary site,w hich is in line with previous data on Lewis X [15] (Figures 2b,c,S 6, and S7). To expand our insight into the presence of secondary sites, we studied 15 N-labeled DC-SIGN CRD by 1 H-15 N HSQC NMR spectroscopy (Figure 2a) [15] and applied computational pocket predictions.T he titration of mannose, am illimolar ligand, led to chemical shift perturbations (CSPs) and reduced resonance intensities for residues close to the primary site,w hich is in line with previous data on Lewis X [15] (Figures 2b,c,S 6, and S7).…”

Uncovering Nonconventional and Conventional Hydrogen Bonds in Oligosaccharides through NMR Experiments and Molecular Modeling: Application to Sialyl Lewis-X

“…Together, the broadening and chemical shift changes are consistent with variable dynamics for the Le X core. 7 After nine days of data collection at 850 MHz (1536 scans per t1 increment) and 37 ˚C, we observed two additional correlations, Fuc{III}H5-Gal{IV}C1 (SNR 11:1) and Fuc{III}H5-GlcNAc{II}C4 (SNR 13:1) (Fig. S3).…”

A Secondary Structural Element in a Wide Range of Fucosylated Glycoepitopes

“…In this context, information on the conformation of the carbohydrate ligandsi nt he unbounda nd bound form is of particulari nterest to understand the entropic contribution to binding.T he trisaccharideL ewis x (Le x ,G alb1,4[Fuca1,3]GlcNAcb)i so ne of the Lewis blood group antigenst hat are widely considered to adopt aw ell-defined structure in solution. [6] Le x is recognized by DC-SIGN [7] and its sialylated version sLe x by E-selectin, [8] as well as the human zona pellucida. [9] Its conformation in solution is virtually identical to the conformations found in the vast majorityo fl ectin complexes,s uggestingt hat the solutionc onformation is identical to the bioactive conformation.Astrongs tackingi nteraction between its Gal and Fuc moiety [10] was attributed to van der Waals contacts, steric hindrance, especially by the adjacent GlcNAc moiety,a sw ell as the exo-anomeric effect.…”