Multivalent lectin-glycan interactions (LGIs) play a key role in cell-cell communication, immune modulation and pathogen infection. As monovalent LGIs are often weak and non-bio-functional, lectins rely on multivalent binding to enhance affinity, either via simultaneous binding or cross-linking. These yield different affinity enhancements and biological consequences, however, their thermodynamic and kinetic bases remain unclear. Additionally, cell membrane lectins are often too flexible to be crystalized for structural studies, therefore, new techniques to reveal glycan-binding-site arrangements are urgently required.

We have recently developed a novel glycan-quantum dot (QD)-based multimodal readout strategy (consisting of FRET, TEM and hydrodynamic size analyses) to probe multivalent LGIs. We found that the tetrameric lectin, DC-SIGN, binds simultaneously to glycan-QDs, while the related DC-SIGNR crosslinks. The latter binding being much weaker than the former. Here, we further employ QD-FRET to investigate their binding thermodynamics and kinetics.

By quantifying DC-SIGN/R-glycan-QD binding affinities at different temperatures via QD-FRET, we reveal that both DC-SIGN/R-glycan-QD bindings are enthalpy driven, with comparable enthalpy terms and a larger entropy penalty for DC-SIGNR binding, responsible for its weaker affinity. Using stopped-flow fluorescence, we find that DC-SIGN-glycan-QD binding is rapid (k_on ~3 x 10⁸ M^-1s^-1) and dissociates slowly (k_off ~2 s^-1), translating to strong nM binding K_ds that are comparable to those obtained from QD-FRET. Using mutagenesis, we also identify that a non-structured segment in DC-SIGN is important for its glycan-QD binding thermodynamics as its removal switches the binding to entropy driven.

To provide structural information, we have developed a novel tagging/double-purification strategy for selectively labelling two of the four tetrameric lectin glycan-binding-domains with a FRET pair. We have then successfully applied single-molecule-FRET to demonstrate measured inter-domain distances that match well to those of DC-SIGNR tetramer models. The distances in DC-SIGN are distinct from those of DC-SIGNR, consistent with their different binding modes to glycan-QDs.

James Hooper

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