Solution structure of the α-subunit of human chorionic gonadotropin

Abstract

The three-dimensional solution structure of the α-subunit in the α, β heterodimeric human chorionic gonadotropin (hCG), deglycosylated with endo-β-N-acetylglucosaminidase-B (dg-α hCG), was determined using 2D homonuclear and 2D heteronuclear 1H, 13C NMR spectroscopy at natural abundance in conjunction with the program package xplor. The distance geometry/simulated annealing protocol was modified to allow for the efficient modelling of the cystine knot motif present in alpha hCG. The protein structure was modelled with 620 interproton distance restraints and the GlcNAc residue linked to Asn78 was modelled with 30 protein-carbohydrate and 3 intraresidual NOEs. The solution structure of dg-alpha hCG is represented by an ensemble of 27 structures. In comparison to the crystal structure of the dimer, the solution structure of free dg-α hCG exhibits: (a) an increased structural disorder (residues 3357); (b) a different backbone conformation near Val76 and Glu77; and (c) a larger flexibility. These differences are caused by the absence of the interactions with the beta-subunit. Consequently, in free dg-α hCG, compared to the intact dimer, the two hairpin loops 2023 and 7074 are arranged differently with respect to each other. The β-GlcNAc(78) is tightly associated with the hydrophobic protein-core in between the beta-hairpins. This conclusion is based on the NOEs from the axial H1, H3, H5 atoms and the N-acetyl protons of β-GlcNAc(78) to the protein-core. The hydrophobic protein-core between the β-hairpins is thereby shielded from the solvent.