Solution structure of the alpha-subunit of human chorionic gonadotropin

The three-dimensional solution structure of the a-subunit in the alpha,beta heterodimeric human chorionic gonadotropin (hCG), deglycosylated with endo -beta-N-acetylglucosaminidase-B (dg-alpha hCG), was determined using 2D hom onuclear and 2D heteronuclear H-1,C-13 NMR spectroscopy at natural abundanc e in conjunction with the program package XPLOR The distance geometry/simul ated annealing protocol was modified to allow for the efficient modelling o f the cystine knot motif present in alpha hCG. The protein structure was mo delled with 620 interproton distance restraints and the GlcNAc residue link ed to Asn78 was modelled with 30 protein-carbohydrate and 3 intraresidual N OEs. The solution structure of dg-alpha hCG is represented by an ensemble o f 27 structures. In comparison to the crystal structure of the dimer, the s olution structure of free dg-alpha hCG exhibits: (a) an increased structura l disorder (residues 33-57); (b) a different backbone conformation near Va1 76 and Glu77; and (c) a larger flexibility. These differences are caused by the absence of the interactions with the beta-subunit. Consequently, in fo e dg-alpha hCG, compared to the intact dimer, the two hairpin loops 20-23 a nd 70-74 are arranged differently with respect to each other. The beta-GlcN Ac(78) is tightly associated with the hydrophobic protein-core in between t he beta-hairpins. This conclusion is based on the NOEs from the axial H1, H 3, H5 atoms and the N-acetyl protons of beta-GLcNAc(78) to the protein-core . The hydrophobic protein-corn between the beta-hairpins is thereby shielde d from the solvent.