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

It has been shown that 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. However, 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 α-helical conformation, we postulate that residues 88-98 are necessary for maintaining the α-helical conformation of the COOH-terminal pentapeptide (99-103) involved in receptor recognition. To test this hypothesis, we have examined the conformational preferences of polypeptide segments from known antigenic regions near the carboxyl terminus of cytochrome c (pigeon, moth, and fly sequences) using conformational energy calculations for peptides in a nonpolar environment. We show here that fragments consisting of residues 88-91 and 94-98 of pigeon, moth and fly cytochrome c have a strong α-helical preference, despite differences in sequence at residues 88-89 (Lys-Ala in pigeon, Ala-Asn in moth, and Pro-Asn in fly). In contrast, the tripeptide 91-93 (Arg-Ala-Asp) has a strong nonhelical preference. Furthermore, the COOH-terminal peptide 99-103 exists as a statistical coil. However, addition of residues 94-98 to residues 99-103 results in a peptide that has a strong preference for α-helix. From these computational results, we predict (i) that fragment 94-103, existing predominantly as an α-helix, should exhibit stimulatory activity and (ii) that the nonhelical peptide 91-93 can be deleted from fragment 88-103 without affecting its antigenicity. Both of these predictions have been borne out by experiments in which the two peptides were synthesized and shown to stimulate a T-cell proliferative response. These results establish a strong correlation between conformation (here, α-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.