Structural Basis of Multivalent Binding to Wheat Germ Agglutinin

Abstract

The inhibition of carbohydrate-protein interactions by tailored multivalent ligands is a powerful strategy for the treatment of many human diseases. Crucial for the success of this approach is an understanding of the molecular mechanisms as to how a binding enhancement of a multivalent ligand is achieved. We have synthesized a series of multivalent N-acetylglucosamine (GlcNAc) derivatives and studied their interaction with the plant lectin wheat germ agglutinin (WGA) by an enzyme-linked lectin assay (ELLA) and X-ray crystallography. The solution conformation of one ligand was determined by NMR spectroscopy. Employing a GlcNAc carbamate motif with alpha-configuration and by systematic variation of the spacer length, we were able to identify divalent ligands with unprecedented high WGA binding potency. The best divalent ligand has an IC(50) value of 9.8 microM (ELLA) corresponding to a relative potency of 2350 (1170 on a valency-corrected basis, i.e., per mol sugar contained) compared to free GlcNAc. X-ray crystallography of the complex of WGA and the second best, closely related divalent ligand explains this activity. Four divalent molecules simultaneously bind to WGA with each ligand bridging adjacent binding sites. This shows for the first time that all eight sugar binding sites of the WGA dimer are simultaneously functional. We also report a tetravalent neoglycopeptide with an IC(50) value of 0.9 microM being 25,500 times higher than that of GlcNAc (6400 times per contained sugar) and the X-ray structure analysis of its complex with glutaraldehyde-cross-linked WGA. Comparison of the crystal structure and the solution NMR structure of the neoglycopeptide as well as results from the ELLA suggest that the conformation of the glycopeptide in solution is already preorganized in a way supporting multivalent binding to the protein. Our findings show that bridging adjacent protein binding sites by multivalent ligands is a valid strategy to find high-affinity protein ligands and that even subtle changes of the linker structure can have a significant impact on the binding affinity.

Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution

“…3B) (Elad et al, 2009;Scheer et al, 2005;Strambio-De-Castillia et al, 2010). Considering the molecular size of a the WGA dimer of ,5 nm (Schwefel et al, 2010) and random fluorophore labeling, the diameter of the central NPC channel appears to be surprisingly consistent. Owing to the large molecular size of gp210 (200 kDa) (Favreau et al, 2001) and the fact that we used IgG antibodies and F(ab9) 2 fragments for indirect labeling with molecular sizes of 8-10 nm, the exact size of the NPC-anchoring gp210 proteins was difficult to determine.…”

Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution

“…1C). The high spatial resolution is due to the large number of photons detected per Alexa 647, ,3.500 photons/frame, and the small size of a WGA dimer, of ,5 nm (Schwefel et al, 2010), used to label the nucleoporins of the central channel. In accordance with this finding, we determined a localization precision of ,6 nm (s.d., in the lateral direction) using localizations of unspecifically bound isolated WGA-Alexa-647 molecules in the sample.…”

Biological Evaluation of Multivalent-Type N-Acetyl-D-Glucosamine (GlcNAc) Conjugates for Wheat Germ Agglutinin (WGA) by the Surface Plasmon Resonance (SPR) Method

“…Although the valency is four, the avidin-biotin scaffold of the ABG complex enhances binding affinity ~ 6380 times than GlcNAc only and ~ 190 times higher than control glycopolymers 1 and 2 and it is assumed that this exceptionally strong effect is to be due to the phenomenon of chelate binding approach [31][32][33]. The distance between binding sites of WGA is 13-14 Å (distances between anomeric oxygen's of two bound GlcNAc residues) [34,35] and the shortest distance between binding sites appears to be as small as 13 Å. For this reason WGA binds strongly to most multivalent analogs that can execute an assured degree of chelation and augment the binding with every valency [36].…”