MUTAGENIC ANALYSIS OF A RECEPTOR CONTACT SITE ON INTERLEUKIN-2 - PREPARATION OF AN IL-2 ANALOG WITH INCREASED POTENCY
MUTAGENIC ANALYSIS OF A RECEPTOR CONTACT SITE ON INTERLEUKIN-2 - PREPARATION OF AN IL-2 ANALOG WITH INCREASED POTENCY
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DOI：
10.1021/bi00187a026
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发表时间：
1994-05-31
期刊：
影响因子：
2.9
通讯作者：
CIARDELLI, TL
中科院分区：
文献类型：
--
作者：
BERNDT, WG;CHANG, DZ;CIARDELLI, TL
白细胞介素-2（IL-2）是由活化的T细胞分泌的133个氨基酸的α-螺旋蛋白。组合盒;诱变用于研究怀疑与中等亲和力IL-2受体相互作用的IL-2的连续五个氨基酸区域的功能作用。构建了IL-2突变体的有限随机文库，其中残基17-21（Leu-Leu-Leu-Asp-Leu）同时突变。蛋白质在大肠杆菌表达系统中产生，并在生物测定中筛选其介导鼠IL-1依赖性细胞系增殖的能力。从超过2600个克隆检查，只有42个表现出显着的活性，证实了该区域的功能的重要性。纯化选择的克隆，并通过生物学和受体结合测定进一步表征。在最近修订的IL-2的2.5埃晶体结构的背景下，这些结果表明以下结论：Asp 20和Leu 21，如它们对突变的敏感性所示，是该区域中功能上更重要的残基，但原因不同。Asp 20是溶剂可及的，并且可能如先前的研究所示起直接受体接触作用。相反，Leu 21完全埋在蛋白质的疏水核心中。发现在该位置的取代，甚至是保守的Leu ->瓦尔取代，扰乱了对活性至关重要的精确的疏水堆积排列，导致。此外，发现筛选中鉴定的一种类似物比野生型蛋白质的效力高2-3倍。
Interleukin-2 (IL-2) is a 133 amino acid alpha-helical protein secreted by activated T-cells. Combinatorial cassette; mutagenesis was used to investigate the functional role of a contiguous five amino acid region of IL-2 suspected to interact with the intermediate-affinity IL-2 receptor. A limited random library of IL-2 mutants was constructed in which residues 17-21 (Leu-Leu-Leu-Asp-Leu) were simultaneously mutated. The proteins were produced in an Escherichia coli expression system and screened in a biological assay for their ability to mediate the proliferation of a murine IL-1-dependent cell line. From the over 2600 clones examined, only 42 exhibited significant activity, confirming the functional importance of this region. Selected clones were purified and further characterized by biological and receptor binding assays. Viewed in the context of the recently revised 2.5-Angstrom crystal structure for IL-2, these results suggest the following conclusions: both Asp20 and Leu21, as shown by their sensitivity to mutation, are the functionally more important residues in this region, but for different reasons. Asp20 is solvent-accessible and likely plays a direct receptor contact role as previous studies have indicated. Leu21, in contrast, is completely buried in the hydrophobic core of the protein. Substitutions at this position, even a conservative Leu --> Val substitution, were found to perturb the precise hydrophobic packing arrangements that are critical for activity, resulting In addition, one of the analogs identified in the screen was found to be 2-3 times more potent than the wild-type protein.