Bagley C J, Phillips J, Cambareri B, Vadas M A, Lopez A F
Division of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5001, Australia.
J Biol Chem. 1996 Dec 13;271(50):31922-8. doi: 10.1074/jbc.271.50.31922.
We have previously reported that, within the first helix of human interleukin (IL)-3, residues Asp21 and Glu22 are important for interaction with the alpha- and beta-chains of the IL-3 receptor, respectively. In order to define more precisely the sites of interaction with the receptor, we have performed molecular modeling of the helical core of IL-3 and single amino acid substitution mutagenesis of residues predicted to lie on the surfaces of the A, C, and D helices. The resulting analogues were characterized for their abilities to stimulate proliferation of TF-l cells and for binding to the high affinity (alpha- and beta-chain; IL-3Ralpha/Rbeta) or low affinity (alpha-chain alone; IL-3Ralpha) IL-3 receptor. We found that in addition to Asp21, residues Ser17, Asn18, and Thr25 within the A helix and Arg108, Phe113, Lys116, and Glu119 within the D helix of IL-3 were important for biological activity. Analysis of their binding characteristics revealed that these analogues were deficient in binding to both the IL-3Ralpha/Rbeta and the IL-3Ralpha forms of the receptor, consistent with a selective impairment of interaction with IL-3Ralpha. Molecular modeling suggests that these eight amino acid residues are adjacent in the tertiary structure, consistent with a discontinuous epitope interacting selectively with IL-3Ralpha. On the other hand, Glu22 of IL-3 was found to interact preferentially with the beta-chain with bulky and positively charged substitutions causing greater than 10,000-fold reduction in biological activity. These results show fundamental differences between IL-3 and granulocyte-macrophage colony-stimulating factor in the structural basis for recognition of their receptors that has implications for the construction of novel analogues and our understanding of receptor activation.
我们先前曾报道，在人白细胞介素（IL）-3的第一个螺旋中，天冬氨酸21和谷氨酸22分别对于与IL-3受体的α链和β链相互作用很重要。为了更精确地确定与受体相互作用的位点，我们对IL-3的螺旋核心进行了分子建模，并对预测位于A、C和D螺旋表面的残基进行了单氨基酸取代诱变。对所得类似物进行了刺激TF-1细胞增殖的能力以及与高亲和力（α链和β链；IL-3Rα/Rβ）或低亲和力（仅α链；IL-3Rα）IL-3受体结合的特性分析。我们发现，除了天冬氨酸21外，IL-3的A螺旋中的丝氨酸17、天冬酰胺18和苏氨酸25以及D螺旋中的精氨酸108、苯丙氨酸113、赖氨酸116和谷氨酸119对于生物学活性也很重要。对它们结合特性的分析表明，这些类似物与受体的IL-3Rα/Rβ和IL-3Rα形式的结合均不足，这与与IL-3Rα相互作用的选择性受损一致。分子建模表明，这八个氨基酸残基在三级结构中相邻，这与一个与IL-3Rα选择性相互作用的不连续表位一致。另一方面，发现IL-3的谷氨酸22优先与β链相互作用，大的带正电荷取代导致生物学活性降低超过10000倍。这些结果表明，IL-3和粒细胞-巨噬细胞集落刺激因子在识别其受体的结构基础上存在根本差异，这对新型类似物的构建以及我们对受体激活的理解具有重要意义。
