Effects of the N-linked glycans on the 3D structure of the free alpha-subunit of human chorionic gonadotropin

To gain insight into intramolecular carbohydrate-protein interactions at th e molecular level, the solution structure of differently deglycosylated var iants of the alpha-subunit of human chorionic gonadotropin have been studie d by NMR spectroscopy. Significant differences in chemical shifts and NOE i ntensities were observed for amino acid residues close to the carbohydrate chain at Asn78 upon deglycosylation beyond Asn78-bound GlcNAc. As no straig htforward strategy is available for the calculation of the NMR structure of intact glycoproteins, a suitable computational protocol had to be develope d. To this end, the X-PLOR carbohydrate force field designed for structure refinement was extended and modified. Furthermore, a computational strategy was devised to facilitate successful protein folding in the presence of ex tended glycans during the simulation. The values for phi and psi dihedral a ngles of the glycosidic linkages of the oligosaccharide core fragments GlcN Ac2(beta 1 -4)GlcNAc1 and Man3(beta 1-4)GlcNAc2 are restricted to a limited range of the broad conformational energy minima accessible for free glycan s. This demonstrates that the protein core affects the dynamic behavior of the glycan at Asn78 by steric hindrance. Reciprocally, the NMR structures i ndicate that the glycan at Asn78 affects the stability of the protein core. The backbone angular order parameters and displacement data of the generat ed conformers display especially for the beta-turn 20-23 a decreased struct ural order upon splitting off the glycan beyond the Asn78-bound GlcNAc. In particular, the Asn-bound GlcNAc shields the protein surface from the hydro philic environment through interaction with predominantly hydrophobic amino acid residues located in both twisted beta-hairpins consisting of residues 10-28 and 59-84.