Consequences of single-chain translation on the structures of two chorionic gonadotropin yoked analogs in alpha-beta and beta-alpha configurations.
Human chorionic gonadotropin (hCG) is a placental-derived heterodimeric glycoprotein hormone, which, through the binding and activation of the LH receptor, rescues the corpus luteum and maintains pregnancy. The three-dimensional structure of hCG is known; however, the relevance of its fold to bioactivity is unclear. Although both subunits (alpha and beta) are required for activity, recent data with single-chain analogs have suggested a diminished role for the cystine knot and an intact heterodimeric interface in binding and receptor activation in vitro. Herein, we report the purification and structural characterization of two yoked (Y) hCG analogs, YhCG1 (beta-alpha) and YhCG3 (alpha-beta). The fusion proteins yielded higher IC50s and EC50s than those of hCG; the maximal hCG-mediated cAMP production, however, was the same. Circular dichroic spectroscopy revealed that the three proteins exhibit distinct far UV circular dichroic spectra, with YhCG1 containing somewhat more secondary structure than YhCG3 and hCG. Limited proteolysis with proteinase K indicated that heterodimeric hCG was much more resistant to cleavage than the single-chain analogs. YhCG1 was more susceptible to proteolysis than YhCG3, and the fragmentation patterns were different in the two proteins. Taken together, the data presented herein provide direct structural evidence for altered three-dimensional conformations in the two single-chain hCG analogs. Thus, the cognate G protein-coupled receptor can recognize and functionally respond to multiple ligand conformations.
Fralish
Gregory B
GB
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229, USA.
Narayan
Prema
P
Puett
David
D
D001483:Base Sequence; D001667:Binding, Competitive; D006063:Chorionic Gonadotropin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism; D019281:Dimerization; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D006063:Chorionic Gonadotropin; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D019286:Endopeptidase K