Functional contributions of noncysteine residues within the cystine knots of human chorionic gonadotropin subunits

Human chorionic gonadotropin (hCG) is a heterodimeric member of a family of cystine knot-containing proteins that contain the consensus sequences Cys- X-1-Gly-X-2-Cys and Cys-X-3-Cys. Previously, we characterized the contribut ions that cystine residues of the hCG subunit cystine knots make in folding , assembly, and bioactivity. Here, we determined the contributions that non cysteine residues make in hCG folding, secretion, and assembly. When the X- 1, X-2, and X-3 residues of hGC-alpha and -beta were substituted by swappin g their respective cystine knot moths, the resulting chimeras appeared to f old correctly and were efficiently secreted. However, assembly of the chime ras with their wild type partner was almost completely abrogated. No single amino acid substitution completely accounted for the assembly inhibition, although the X-2 residue made the greatest individual contribution. Analysi s by tryptic mapping, high performance liquid chromatography, and SDS-polya crylamide gel electrophoresis revealed that substitution of the central Gly in the Cys-X-1-Gly-X-2-Cys sequence of either the alpha- or beta -subunit cystine knot resulted in non-native disulfide bond formation and subunit mi sfolding. This occurred even when the most conservative change possible (Gl y --> Ala) was made. From these studies we conclude that all three "X" resi dues within the hCG cystine knots are collectively, but not individually, r equired for the formation of assembly-competent hCG subunits and that the i nvariant Gly residue is required for efficient cystine knot formation and s ubunit folding.