A cryptic pocket allosterically modulates oligosaccharide binding to DC-SIGN

DC-SIGN is a C-type lectin receptor expressed on antigen-presenting cells, crucial for pathogen recognition and immune modulation. Here, we identify and characterize a previously unrecognized cryptic allosteric pocket in DC-SIGN using molecular dynamics simulations, NMR spectroscopy, cryogenic electron microscopy and biochemical assays. Rotation of the gatekeeper residue M270 exposes the pocket whose occupancy modulates glycan binding. Mutations M270F and T314A mimic the occupied and unoccupied states of this pocket, respectively, shifting the conformational equilibrium of α-helix 2 and altering oligosaccharide affinity via the extended carbohydrate binding site. While Ca²⁺ coordination at the canonical binding site remains unaffected, our data reveal a complex interplay between the Ca²⁺ binding sites and the canonical and extended glycan binding surfaces. These findings uncover a hierarchical allosteric mechanism that enables selective tuning of glycan affinity and suggest the cryptic pocket as a novel target for drug discovery in C-type lectins.