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Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein – Ligand Interactions

View Article: PubMed Central - PubMed

ABSTRACT

A highly efficient cap‐exchange approach for preparing compact, dense polyvalent mannose‐capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC‐SIGN and DC‐SIGNR (collectively termed as DC‐SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC‐SIGN, but not its closely related receptor DC‐SIGNR, which is further confirmed by its specific blocking of DC‐SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC‐SIGN binds more efficiently to densely packed mannosides. A FRET‐based thermodynamic study reveals that the binding is enthalpy‐driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein–ligand interactions.

No MeSH data available.


Schematics of the different DC‐SIGN/R‐QD‐Man multi‐valent binding. A) Schematic structure of a DC‐SIGN/R subunit. B) The uprightly facing DC‐SIGN CRDs readily bind to multiple mannoses on the QD, resulting in strong multivalent binding. C) The sideway pointing CRDs in DC‐SIGNR cannot bind to multiple sugars on the QD simultaneously, leading to weak/minimal binding.
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anie201600593-fig-0004: Schematics of the different DC‐SIGN/R‐QD‐Man multi‐valent binding. A) Schematic structure of a DC‐SIGN/R subunit. B) The uprightly facing DC‐SIGN CRDs readily bind to multiple mannoses on the QD, resulting in strong multivalent binding. C) The sideway pointing CRDs in DC‐SIGNR cannot bind to multiple sugars on the QD simultaneously, leading to weak/minimal binding.

Mentions: The apparent KDs for DC‐SIGN‐QD binding were all in the high nm range (Table 1), >5000‐fold tighter than individual mannose–CRD binding (KD=3.5 mm),9 indicating that multivalent binding greatly enhanced the binding affinity. Because the mannose moieties are covalently coupled to a solid, non‐deformable and spherical QD core, only receptors having CRDs that face in the same direction are able to bind multivalently to the QD. The minimal DC‐SIGNR‐QD binding revealed here implies an inability to form effective multivalent binding. Based on their distinct QD‐binding properties, we propose that the CRDs are facing upwardly along the coiled‐coil axes in DC‐SIGN (hence readily accessible to multivalent binding to the QD), but sideways in DC‐SIGNR (hence unavailable to bind the QD multivalently, Figure 4). Such structural models agree well with those proposed from small‐angle X‐ray scattering studies.13 The different CRD arrangement and accessibility in DC‐SIGN/R may account for their distinct viral binding/transmission properties. It also correlates well with the biological roles: the high accessibility of DC‐SIGN should enable rapid antigen capture to trigger the immune response as required for an antigen‐presenting dendritic cell surface endocytic receptor.11, 14 Whereas the endothelial cell surface adhesion receptor DC‐SIGNR11 may only recognize specific, spatial and orientation‐matched multivalent glycans.


Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein – Ligand Interactions
Schematics of the different DC‐SIGN/R‐QD‐Man multi‐valent binding. A) Schematic structure of a DC‐SIGN/R subunit. B) The uprightly facing DC‐SIGN CRDs readily bind to multiple mannoses on the QD, resulting in strong multivalent binding. C) The sideway pointing CRDs in DC‐SIGNR cannot bind to multiple sugars on the QD simultaneously, leading to weak/minimal binding.
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979658&req=5

anie201600593-fig-0004: Schematics of the different DC‐SIGN/R‐QD‐Man multi‐valent binding. A) Schematic structure of a DC‐SIGN/R subunit. B) The uprightly facing DC‐SIGN CRDs readily bind to multiple mannoses on the QD, resulting in strong multivalent binding. C) The sideway pointing CRDs in DC‐SIGNR cannot bind to multiple sugars on the QD simultaneously, leading to weak/minimal binding.
Mentions: The apparent KDs for DC‐SIGN‐QD binding were all in the high nm range (Table 1), >5000‐fold tighter than individual mannose–CRD binding (KD=3.5 mm),9 indicating that multivalent binding greatly enhanced the binding affinity. Because the mannose moieties are covalently coupled to a solid, non‐deformable and spherical QD core, only receptors having CRDs that face in the same direction are able to bind multivalently to the QD. The minimal DC‐SIGNR‐QD binding revealed here implies an inability to form effective multivalent binding. Based on their distinct QD‐binding properties, we propose that the CRDs are facing upwardly along the coiled‐coil axes in DC‐SIGN (hence readily accessible to multivalent binding to the QD), but sideways in DC‐SIGNR (hence unavailable to bind the QD multivalently, Figure 4). Such structural models agree well with those proposed from small‐angle X‐ray scattering studies.13 The different CRD arrangement and accessibility in DC‐SIGN/R may account for their distinct viral binding/transmission properties. It also correlates well with the biological roles: the high accessibility of DC‐SIGN should enable rapid antigen capture to trigger the immune response as required for an antigen‐presenting dendritic cell surface endocytic receptor.11, 14 Whereas the endothelial cell surface adhesion receptor DC‐SIGNR11 may only recognize specific, spatial and orientation‐matched multivalent glycans.

View Article: PubMed Central - PubMed

ABSTRACT

A highly efficient cap‐exchange approach for preparing compact, dense polyvalent mannose‐capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC‐SIGN and DC‐SIGNR (collectively termed as DC‐SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC‐SIGN, but not its closely related receptor DC‐SIGNR, which is further confirmed by its specific blocking of DC‐SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC‐SIGN binds more efficiently to densely packed mannosides. A FRET‐based thermodynamic study reveals that the binding is enthalpy‐driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein–ligand interactions.

No MeSH data available.