Palla E, Bensi G, Solito E, Buonamassa D T, Fassina G, Raugei G, Spano F, Galeotti C, Mora M, Domenighini M
Department of Molecular Biology, Immunobiology Research Institute Siena, Italy.
J Biol Chem. 1993 Jun 25;268(18):13486-92.
By computer analysis of the amino acid sequence of human interleukin-1 beta (IL-1 beta) and of the human type I IL-1 receptor (IL-1RI), we have identified two hydropathically complementary peptides (Fassina, G., Roller, P. P., Olson, A. D., Thorgeirsson, S. S., and Omichinski, J. G. (1989) J. Biol. Chem. 264, 11252-11257) capable of binding to each other. The sequence of the IL-1 beta peptide corresponds to that of residues 88-99 (loop 7 of the crystal structure of mature IL-1 beta) of mature IL-1 beta, one of the exposed and highly charged regions of the molecule. The substitution of this loop with an amino acid sequence of the same length but different hydropathic profile generates a mutant with drastically reduced binding activity to IL-1RI. In contrast, the binding affinity to the type II IL-1R (IL-1RII) is the same as that of wild type IL-1 beta. The results show that 1) loop 7 is part of the binding site of IL-1 beta to IL-1RI, but not to IL-1RII. 2) The structure of the mutant protein is not grossly altered except locally at the position of the substituted loop. 3) The substitution of amino acids by site-directed mutagenesis of the loop 7 region generates mutants with binding affinity constants slightly lower than that of wild type IL-1 beta and not comparable to that of the loop substitution analogue. 4. All mutants analyzed, including the loop substitutions, are biologically active, confirming the structural integrity of the proteins. We propose a binding site in which the cooperation of several low energy bonds extended over a wide area results in a high affinity complex between IL-1 and the type I receptor.
通过对人白细胞介素-1β（IL-1β）和人I型IL-1受体（IL-1RI）的氨基酸序列进行计算机分析，我们鉴定出了两条具有亲水性互补的肽段（法西纳，G.，罗勒，P.P.，奥尔森，A.D.，索尔吉尔松，S.S.，和奥米奇尼斯基，J.G.（1989年）《生物化学杂志》264卷，11252 - 11257页），它们能够相互结合。IL-1β肽段的序列对应于成熟IL-1β的88 - 99位残基（成熟IL-1β晶体结构的环7），这是该分子暴露且电荷高度集中的区域之一。用长度相同但亲水性分布不同的氨基酸序列替换这个环，会产生一个与IL-1RI结合活性大幅降低的突变体。相比之下，其对II型IL-1受体（IL-1RII）的结合亲和力与野生型IL-1β相同。结果表明：1）环7是IL-1β与IL-1RI结合位点的一部分，但不是与IL-1RII结合位点的一部分。2）突变蛋白的结构除了在被替换环的位置局部有所改变外，整体没有明显变化。3）通过对环7区域进行定点诱变替换氨基酸，产生的突变体结合亲和常数略低于野生型IL-1β，且与环替换类似物的亲和常数不可比。4）所有分析的突变体，包括环替换突变体，都具有生物活性，证实了蛋白质的结构完整性。我们提出了一个结合位点，其中几个低能键在广泛区域内协同作用，导致IL-1与I型受体之间形成高亲和力复合物。
