Preferred conformations and dynamics of five core structures of mucin type O-glycans determined by NMR spectroscopy and force field calculations

Glycosyltransferases acting on O-glycans have been shown to exhibit distinct specificity for the carbohydrate and the peptide moiety of their substrates. As an approach to study the 3-dimensional interactions between enzymes and O-glycan substrates, we determined the preferred conformations of five oligosaccharide-core structures of mucin type glycoproteins by NMR spectroscopy and by static and dynamic force field calculations. Seven oligosaccharides, representing five basic core structures, were investigated: Galβ(1-3)GalNAcαBzl (1, core 1), GlcNAcβ(1-6)[Galβ(1-3)]GalNAcαBzl (2, core 2), GlcNAcβ(1-3)GalNacαBzl (3, core 3), GlcNAcβ(1-6)[GlcNAcβ(1-3)]GalNAcαBzl (4, core 4), GlcNAcβ(1-6)GalNAcαBzl (5, core 6), the elongated core 2, Galβ(1-4)GlcNAcβ(1-6)[Galβ(1-3)]GalNAcαpNp (6) and GalNAcα-Bzl (7). The dynamic behaviour of the molecules was studied by Metropolis Monte Carlo (MMC) simulations. Experimental coupling constants, chemical shift changes, and NOEs were compared with results from static energy minimizations and dynamic MMC simulations and show a good agreement. MMC simulations show that the (1-6) linkage is much more flexible than the (1-3) or the (1-4) linkages. The preferred conformations of the disaccharides (1) and (3) show only slight differences due to the additional N-acetyl group in (3). The conformational equilibrium of β(1-3) glycosidic bonds of 1 and 3 was not affected by attaching a β(1-6) linked GlcNAc unit to the GalNAc residue in 2 and 4. However, experimental and theoretical data show that the β(1-6) linkages of the trisaccharides 2 and 4, which carry an additional β(1-3) linked glycosyl residue, change their preferred conformations when compared with (5). The 6-branch also shows significant interactions with the benzyl aglycon altering the preferred conformation of the hydroxymethyl group of the GalNAc to a higher proportion of the gt conformer. The (1-6) linkage of 2, 4, and 6 can have two different families of conformations of which the lower energy state is populated only to about 20% of the time whereas the other state with a relative enthalpy of ≈4 kcal mol^-1 is populated to 80%. This fact demonstrates that the two conformational states have different entropy contents. Entropy is implicitly included in MMC simulations but cannot be derived from energy minimizations.