Multivalent lectin−glycan interactions (MLGIs) are widespread and vital for biology. Their binding biophysical and structural details are thus highly valuable, not only for the understanding of binding affinity and specificity mechanisms but also for guiding the design of multivalent therapeutics against specific MLGIs. However, effective techniques that can reveal all such details remain unavailable. We have recently developed polyvalent glycan quantum dots (glycan-QDs) as a new probe for MLGIs. Using a pair of closely related tetrameric viral-binding lectins, DC-SIGN and DC-SIGNR, as model examples, we have revealed and quantified their large affinity differences in glycan-QD binding are due to distinct binding modes: with simultaneous binding for DC-SIGN and cross-linking for DC-SIGNR. Herein, we further extend the capacity of the glycan-QD probes by investigating the correlation between binding mode and binding thermodynamics and kinetics and further probing a structural basis of their binding nature. We reveal that while both lectins' binding with glycan-QDs is enthalpy driven with similar binding enthalpy changes, DC-SIGN pays a lower binding entropy penalty, resulting in a higher affinity than DC-SIGNR. We then show that DC-SIGN binding gives a single second-order k on rate, whereas DC-SIGNR gives a rapid initial binding followed by a much slower secondary interaction. We further identify a structural element in DC-SIGN, absent in DC-SIGNR, that plays an important role in maintaining DC-SIGN's MLGI character. Its removal switches the binding from being enthalpically to entropically driven and gives mixed binding modes containing both simultaneous and cross-linking binding behavior, without markedly affecting the overall binding affinity and kinetics

Multivalent lectin-glycan interactions (MLGIs) are widespread in biology and hold the key to many therapeutic applications. However, the underlying structural and biophysical mechanisms for many MLGIs remain poorly understood, limiting our ability to design glycoconjugates to potently target specific MLGIs for therapeutic intervention. Glycosylated nanoparticles have emerged as a powerful biophysical probe for MLGIs, although how nanoparticle shape affects the MLGI molecular mechanisms remains largely unexplored. Herein, we have prepared fluorescent quantum nanorods (QRs), densely coated with α-1,2-manno-biose ligands (QR-DiMan), as multifunctional probes to investigate how scaffold geometry affects the MLGIs of a pair of closely related, tetrameric viral receptors, DC-SIGN and DC-SIGNR. We have previously shown that a DiMan-capped spherical quantum dot (QD-DiMan) gives weak cross-linking interactions with DC-SIGNR but strong simultaneous binding with DC-SIGN. Against the elongated QR-DiMan, DC-SIGN retains similarly strong simultaneous binding of all four binding sites with a single QR-DiMan (apparent K d ≈ 0.5 nM, ∼1.8 million-fold stronger than the corresponding monovalent binding), while DC-SIGNR gives both weak cross-linking and strong individual binding interactions, resulting in a larger binding affinity enhancement than that with QD-DiMan. S/TEM analysis of QR-DiMan-lectin assemblies reveals that DC-SIGNR's different binding modes arise from the different nanosurface curvatures of the QR scaffold. The glycan display at the spherical ends presents too high a steric barrier for DC-SIGNR to bind with all four binding sites; thus, it cross-links between two QR-DiMan to maximize binding multivalency, whereas the more planar character of the cylindrical center allows the glycans to bridge all binding sites in DC-SIGNR. This work thus establishes glycosylated QRs as a powerful biophysical probe for MLGIs not only to provide quantitative binding affinities and binding modes but also to demonstrate the specificity of multivalent lectins in discriminating different glycan displays in solution, dictated by the scaffold curvature.