NMR studies of the free α subunit of human chorionic gonadotropin. Structural influences of N-glycosylation and the β subunit on the conformation of the α subunit

Abstract

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone that is involved in the maintenance of the corpus luteum in early pregnancy. Glycosylation at Am52 of its α subunit (α hCG) is essential for signal transduction, whereas the N-glycan at Am78 stabilizes the structure of the protein. In this study, an almost complete 1H-NMR and a partial 13C-NMR spectral assignment for the amino acids and the N-glycans of αhCG and of an enzymatically deglycosylated form, which had a single GlcNAc residue at each of its two glycosylation sites, has been achieved. The secondary structure of hCG in solution, which was determined based on NOE data, is partially similar to that of the α subunit in the crystal structure of hCG, but large structural differences are found for amino acid residues 3358. In the crystal structure of hCG, residues 3337 and 5458 of the a subunit are part of an intersubunit seven-stranded β-barrel and residues 4147 constitute a 310-helix. In contrast, in free α hCG in solution, amino acids 3358 are part of a large disordered loop, indicating that in intact hCG interactions with the β subunit of hCG stabilize the conformation of the α subunit. The NMR data of alpha hCG and its deglycosylated counterpart are very similar, indicating that removal of carbohydrate residues other than GlcNAc-1 does not notably affect the conformation of the protein part. However, numerous 1H-NOES between the GlcNAc-1 residue at Am78 and several amino acid residues show that this GlcNAc residue is tightly packed against the protein, being an integral part of the structure of the α subunit. 1H-NOES across the glycosidic linkages of the glycan, resonance-line widths, and 1H and 13C chemical shifts of the other monosaccharides suggest that the remainder of the glycans at Asn78, and the glycans at Asn52 are largely extended in solution.