Probing Thermodynamics, Kinetics and Structural Details of Multivalent Lectin-Glycan Interactions by Quantum Dot-FRET

31 March 2022, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Multivalent lectin-glycan interactions (MLGIs) are widely employed for bio- recognition and discrimination, but they are also exploited by pathogens to infect host cells. Their biophysical details (e.g. thermodynamics, kinetics, binding modes and binding site orientation) are thus highly valuable, not only for elucidating the underlying mechanisms, but also for guiding the design of multivalent therapeutics against specific MLGIs. However, these details are not readily available due to the limitations of conventional biophysical techniques in probing such complex, flexible interactions. We have recently established densely glycosylated quantum dots (glycan-QDs) as novel structural probes for MLGIs. Using a pair of important, almost identical tetrameric lectins, DC-SIGN and DC-SIGNR, as the model lectins, we have shown that glycan-QDs can not only provide quantitative binding affinities but also dissect their distinct binding modes: DC-SIGN binds simultaneously with one glycan-QD whereas DC-SIGNR inter-cross-links. Herein, we further extend the capacity of the glycan-QD probes to investigate how binding mode affects the binding thermodynamics and kinetics, and probe a structural basis of their binding nature. We show that, while both lectin-glycan-QD interactions are enthalpy driven, with similar binding enthalpy changes (~4 times that of monovalent binding measured by ITC), DC-SIGN binding pays a lesser entropy penalty than DC-SIGNR, giving rise to a stronger affinity. We also reveal that a short C-terminal segment at the flexible junction between the tetramerization domain and glycan binding domain in DC-SIGN, absent in DC-SIGNR, plays a critical role in maintaining DC-SIGN’s glycan-QD binding properties: its removal leads to an entirely different binding enthalpy and entropy profile, despite maintaining the same binding mode. Furthermore, we show that the simultaneous lectin-glycan-QD binding partners give single 2nd-order kon rates which rapidly reach saturation, whereas cross-linking partners give two distinct on-rates: a rapid initial association step, followed by a much slower secondary interaction. Together, our work have established glycan-QDs as a powerful new biophysical platform for solution-based MLGI studies which can provide a wide range of important biophysical parameters.


Multivalent lectin-glycan interaction
quantum dot
Lectin structure and function

Supplementary materials

Probing Thermodynamics, Kinetics and Structural Details of Multi-valent Lectin-Glycan Interactions by Quantum Dot-FRET
Supporting information for Thermodynamic manu


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