Correlation between the conformation of cytochrome c peptides and their stimulatory activity in a T-lymphocyte proliferation assay.

Abstract

The minimal-length peptide having full stimulatory activity for pigeon cytochrome c-primed T cells from B10.A mice is composed of residues 88-103 of the moth (or 87-104 of the pigeon) sequence. To date, only residues 99-103(104) have been shown to be involved in contacting the T cell receptor or the macrophage Ia molecule. Because the X-ray structure of tuna cytochrome c, and prior calculations on many homologous cytochrome c proteins, showed that segment 88-103(104) exists in the .alpha.-helical conformation, it is postulate that residues 88-98 are necessary for maintaining the .alpha.-helical conformation of the COOH-terminal pentapeptide (99-103) involved in receptor recognition. To test this hypothesis, the conformational preferences of polypeptide segments from known antigenic regions near the carboxyl terminus of cytochrome c (pigeon, moth and fly sequences) were examined using conformational energy calculations for peptides in a nonpolar environment. Fragments consisting of residues 88-91 and 94-98 of pigeon, moth and fly cytochrome c have a strong .alpha.-helical preference, despite differences in sequence at residues 88-89 (Lys-Ala in pigeon, Ala-Asn in moth, and Pro-Asn in fly). The tripeptide 91-93 (Arg-Ala-Asp) has a strong nonhelical preference. The COOH-terminal peptide 99-103 exists as a statistical coil. Addition of residues 94-98 to residues 99-103 results in a peptide that has a strong preference for .alpha.-helix. From these computational results, it is predicted; that fragment 94-103, existing predominantly as an .alpha.-helix, should exhibit stimulatory activity; and that the nonhelical peptide 91-93 can be deleted from fragment 88-103 without affecting its antigenicity. Both of these predictions were borne out by experiments in which the 2 peptides were synthesized and stimulated a T cell proliferative response. The results establish a strong correlation between conformation (here, .alpha.-helix) and biological activity and suggest that T cell activation is sensitive to the organized backbone structure that the antigen adopts in the nonpolar environment of the macrophage membrane or in the combining site of the T cell receptor.