Pincus M R, Gerewitz F, Schwartz R H, Scheraga H A
Proc Natl Acad Sci U S A. 1983 Jun;80(11):3297-300. doi: 10.1073/pnas.80.11.3297.
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 alpha-helical conformation, we 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, 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 alpha-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 alpha-helix. From these computational results, we predict (i) that fragment 94-103, existing predominantly as an alpha-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, 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.
已表明，对来自B10.A小鼠的鸽细胞色素c致敏的T细胞具有完全刺激活性的最短长度肽由蛾序列的88 - 103位残基（或鸽序列的87 - 104位残基）组成。然而，迄今为止，仅99 - 103（104）位残基被证明参与与T细胞受体或巨噬细胞Ia分子的接触。因为金枪鱼细胞色素c的x射线结构以及之前对许多同源细胞色素c蛋白的计算表明，88 - 103（104）片段以α螺旋构象存在，我们推测88 - 98位残基对于维持参与受体识别的COOH末端五肽（99 - 103）的α螺旋构象是必需的。为了验证这一假设，我们使用非极性环境中肽的构象能量计算，研究了细胞色素c（鸽、蛾和果蝇序列）羧基末端附近已知抗原区域的多肽片段的构象偏好。我们在此表明，由鸽、蛾和果蝇细胞色素c的88 - 91位残基和94 - 98位残基组成的片段具有很强的α螺旋偏好，尽管88 - 89位残基的序列存在差异（鸽中为Lys - Ala，蛾中为Ala - Asn，果蝇中为Pro - Asn）。相反，三肽91 - 93（Arg - Ala - Asp）具有很强的非螺旋偏好。此外，COOH末端肽99 - 103以统计卷曲形式存在。然而，将94 - 98位残基添加到99 - 103位残基上会产生一种对α螺旋有强烈偏好的肽。从这些计算结果中，我们预测：（i）主要以α螺旋形式存在的94 - 103片段应具有刺激活性；（ii）非螺旋肽91 - 93可以从88 - 103片段中删除而不影响其抗原性。这两个预测都已通过实验得到证实，在实验中合成了这两种肽并显示它们能刺激T细胞增殖反应。这些结果在构象（此处为α螺旋）与生物活性之间建立了很强的相关性，并表明T细胞活化对抗原在巨噬细胞膜的非极性环境或T细胞受体结合位点所采用的有组织的主链结构敏感。
