Mechanism of multivalent carbohydrate-protein interactions studied by EPR spectroscopy.

The interaction of multivalent carbohydrate derivatives with carbohydrate-binding proteins (lectins) is frequently observed in biological systems, and their inhibition by tailored multivalent ligands is a powerful strategy for the treatment of many human diseases. fli Compared to monovalent interactions, multivalency can lead to significantly increased binding affinities and specificities. Several mechanisms have been suggested to be responsible for the observed binding enhancements. fl f. 1 Among them is the spanning of adjacent binding sites by the multivalent ligand (chelate effect)[31 to which the highest contribution to enhanced binding affinity is attributed. Recently, we were able to characterize chelating binding of multivalent N-acetylglucosamine (GlcNAc) derivatives to wheat germ agglutinin (WGA) by X-ray crystallography.l4] Since it is well established that the structure of biomolecules determined by X-ray crystallography is not necessarily identical to their solution structure[SI and, furthermore, binding mechanisms in a densely packed crystal and in solution may differ, a method to determine the binding mode of multivalent interactions in solution is desirable. Here, we describe the application of electron paramagnetic resonance (EPR) spectroscopy of spin-labeled ligands for this purpose and provide structural evidence for chelating binding of a multivalent GlcNAc derivative to WGA in solution. WGA is a plant lectin that forms a 36 kDa stable homodimer with a twofold symmetry axis and is specific for terminal N-acetylneuraminic acid and GlcNAc.[6] WGA contains eight binding sites, termed A1, BIC2, CIB2, DIA2, A2, B2Cl, C2Bl, and D2A1.171 We could show by Xray crystallography that four molecules of divalent ligand 1 (Scheme 1) simultaneously bind to all e ight GlcNAc binding sites of the WGA dimer with each ligand bridging pairs of adjacent binding sites, specifically, BIC2-C2B1, B2CI-CIB2, AI-D2Al , and A2Dl A2.141 This structure explains the high binding affinity of 1 towards WGA (lC50 = 57 J.LM)141 that had