Primary and secondary structural determinants in the receptor binding sequence .beta.-(38-57) from human luteinizing hormone

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Abstract
The intercysteine "loop" sequence 38-57 in the .beta. subunit has been shown to be a determinant for expression of biological activity in human lutropin (hLH) and choriogonadotropin (hCG) [Keutmann, H. T., Charlesworth, M. C., Mason, K. A., Ostrea, T., Johnson, L., and Ryan, R. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2038]. Together with other sequences, the 38-57 region may contribute to a multicomponent receptor binding domain in hLH/hCG. Because the structural features influencing activity in this important region are not easy to evaluate in the full-length subunit, we have used analogues of hLH.beta.-(38-57) prepared by solid-phase synthesis. The peptides were tested for inhibition of 125I-labeled hCG binding to rat ovarian membrane receptors. Secondary structure was analyzed by circular dichroism (CD) and by reactivity with antibodies to the native 38-57 peptide. An analogue lacking the 38-57 disulfide linkage retained 20% receptor binding and full immunoreactivity. "Far"-ultraviolet CD profiles were essentially identical with those of the disulfide-intact peptide, a transition from 10% to 30% .alpha.-helix in 90% trifluoroethanol was characteristic of both. The peptide thus appears not to require the disulfide bridge to retain a looped conformation with amphipathic secondary structure. An essential positive charge at position 43 was shown by complete loss of activity upon substitution of Asp or Ala for the Arg found in all known species of LH. Other analogues showed a requirement for a neutral residue at position 47, also highly conserved. One or more of the prolines at 50, 51, and 53 may confer a bend to help maintain the loop conformation. Substitution of alanine for individual prolines was tolerated, with binding constants comparable to that of the native peptide [(2.5-3.5) .times. 10-5 M]. The double substitution of Ala for Pro at 50 and 51 altered the binding response and changed the physical characteristics toward a more open loop with a marked tendency toward dimerization. These results indicate that the 38-57 sequence is a relatively rigid and structurally autonomous region, not merely a series of residues constrained passively into a loop by a disulfide linkage. It includes segments of ordered structure, probably including both amphipathic helical and turn sequences. Evidence from studies of other hormones suggests that this region may be important to binding and specificity in the glycoprotein hormones as a group.