APPLICATION OF HOMONUCLEAR 3D NMR EXPERIMENTS AND 1D ANALOGS TO STUDYTHE CONFORMATION OF SIALYL LEWIS(X) BOUND TO E-SELECTIN

The conformation of the sialyl Lewis(x) tetrasaccharide bound to E-sel ectin was previously determined from transfer NOE (trNOE) experiments in conjunction with a distance-geometry analysis. However, the orienta tion of the tetrasaccharide ligand in the binding site of E-selectin i s still unknown. It can be predicted that the accurate quantitative an alysis of all trNOEs, including those originating from spin diffusion, is one key to analyze the orientation of sialyl Lewis(x) in the bindi ng pocket of E-selectin. Therefore, we applied homonuclear 3D NMR expe riments and 1D analogs to obtain trNOEs that could not unambiguously b e assigned from previous 2D trNOESY spectra, due to severe resonance-s ignal overlap. A 3D TOCSY-trNOESY experiment, a 1D TOCSY-trNOESY exper iment, and a 1D trNOESY-TOCSY experiment of the sialyl Lewis(x)/E-sele ctin complex furnished new interglycosidic trNOEs and provided additio nal information for the interpretation of trNOEs that have been descri bed before. A 2D trROESY spectrum of the sialyl Lewis(x)/E-selectin co mplex allowed one to identify the amount of spin-diffusion contributio ns to trNOEs, Finally, an unambiguous assignment of all trNOEs, and an analysis of spin-diffusion pathways, was obtained, creating a basis f or a quantitative analysis of trNOEs in the sialyl Lewis(x)/E-selectin complex.