Hill C M, Liu A, Marshall K W, Mayer J, Jorgensen B, Yuan B, Cubbon R M, Nichols E A, Wicker L S, Rothbard J B
ImmuLogic Pharmaceutical Corp., Palo Alto, CA 94304.
J Immunol. 1994 Mar 15;152(6):2890-8.
The individual amino acid contacts responsible for peptide binding to DRB10101 and/or DRB10401 were defined using a quantitative binding assay. The differential contribution of each amino acid in two well studied T cell determinants, HA307-319 and RMBP 90-102, was delineated by comparing the IC50 values of analogues of varying length. This analysis confirmed the importance of a hydrophobic amino acid located near the amino-terminus for binding to both alleles and revealed that the contribution of the carboxyl-terminal amino acids differed between DRB10101 and DRB10401. Taking advantage of previous experiments demonstrating that all of the residues could be replaced with alanine, with the exception of the key hydrophobic amino acid, simplified analogues composed of polyalanines were used to prove 1) optimal binding depended on the position of the hydrophobic side chain relative to the amino- and carboxyl-termini; 2) aromatic amino acids were superior to aliphatic side chains at this position; and 3) a significant amount of free energy of binding arises from hydrogen bonding between the class II binding site and the amide bonds of the ligand. The role of each carbonyl and amide nitrogen was measured by assaying analogues containing reduced peptide bonds or N-methyl amino acids. Serine, but not glycine, could be used as a framework amino acid for peptide ligands, indicating that the beneficial aspects of these simplified structures was the combination of retaining the correct orientation of the peptide bonds, the restriction of the conformational freedom by limiting the possible phi/psi angles of the peptide, and avoidance of deleterious side-chain contacts. Collectively, these data were consistent with the peptide binding in a nonrepeating conformation with the vast majority of the free energy of binding arising from hydrogen bonds with the peptide backbone and a single, key hydrophobic side chain interacting in a conserved pocket in both DRB10101 and DRB10401.
利用定量结合试验确定了负责肽与DRB10101和/或DRB10401结合的单个氨基酸接触点。通过比较不同长度类似物的IC50值，描绘了两个深入研究的T细胞决定簇HA307 - 319和RMBP 90 - 102中每个氨基酸的不同贡献。该分析证实了氨基末端附近的疏水氨基酸对与两个等位基因结合的重要性，并揭示了DRB10101和DRB10401之间羧基末端氨基酸的贡献有所不同。利用先前的实验表明，除关键疏水氨基酸外，所有残基都可用丙氨酸替代，使用由聚丙氨酸组成的简化类似物来证明：1）最佳结合取决于疏水侧链相对于氨基和羧基末端的位置；2）在该位置芳香族氨基酸优于脂肪族侧链；3）大量的结合自由能来自II类结合位点与配体酰胺键之间的氢键。通过检测含有减少肽键或N-甲基氨基酸的类似物来测量每个羰基和酰胺氮的作用。丝氨酸而非甘氨酸可作为肽配体的骨架氨基酸，这表明这些简化结构的有益之处在于保留肽键正确取向、通过限制肽的可能φ/ψ角来限制构象自由度以及避免有害的侧链接触的组合。总体而言，这些数据与肽以非重复构象结合一致，其中绝大多数结合自由能来自与肽主链的氢键以及单个关键疏水侧链在DRB10101和DRB10401的保守口袋中相互作用。
