Mukai Yohei, Shibata Hiroko, Nakamura Teruya, Yoshioka Yasuo, Abe Yasuhiro, Nomura Tetsuya, Taniai Madoka, Ohta Tsunetaka, Ikemizu Shinji, Nakagawa Shinsaku, Tsunoda Shin-ichi, Kamada Haruhiko, Yamagata Yuriko, Tsutsumi Yasuo
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
J Mol Biol. 2009 Jan 30;385(4):1221-9. doi: 10.1016/j.jmb.2008.11.053. Epub 2008 Dec 6.
Tumor necrosis factor (TNF) is an important cytokine that suppresses carcinogenesis and excludes infectious pathogens to maintain homeostasis. TNF activates its two receptors [TNF receptor (TNFR) 1 and TNFR2], but the contribution of each receptor to various host defense functions and immunologic surveillance is not yet clear. Here, we used phage display techniques to generate receptor-selective TNF mutants that activate only one TNFR. These TNF mutants will be useful in the functional analysis of TNFR. Six amino acids in the receptor binding interface (near TNF residues 30, 80, and 140) were randomly mutated by polymerase chain reaction. Two phage libraries comprising over 5 million TNF mutants were constructed. By selecting the mutants without affinity for TNFR1 or TNFR2, we successfully isolated 4 TNFR2-selective candidates and 16 TNFR1-selective candidates, respectively. The TNFR1-selective candidates were highly mutated near residue 30, whereas TNFR2-selective candidates were highly mutated near residue 140, although both had conserved sequences near residues 140 and 30, respectively. This finding suggested that the phage display technique was suitable for identifying important regions for the TNF interaction with TNFR1 and TNFR2. Purified clone R1-6, a TNFR1-selective candidate, remained fully bioactive and had full affinity for TNFR1 without activating TNFR2, indicating the usefulness of the R1-6 TNF mutant in analyzing TNFR1 receptor function. To further elucidate the receptor selectivity of R1-6, we examined the structure of R1-6 by X-ray crystallography. The results suggested that R31A and R32G mutations strongly influenced electrostatic interaction with TNFR2, and that L29K mutation contributed to the binding of R1-6 to TNFR1. This phage display technique can be used to efficiently construct functional mutants for analysis of the TNF structure-function relationship, which might facilitate in silico drug design based on receptor selectivity.
肿瘤坏死因子（TNF）是一种重要的细胞因子，可抑制肿瘤发生并排除感染性病原体以维持体内平衡。TNF激活其两种受体[TNF受体（TNFR）1和TNFR2]，但每种受体对各种宿主防御功能和免疫监视的贡献尚不清楚。在此，我们使用噬菌体展示技术生成仅激活一种TNFR的受体选择性TNF突变体。这些TNF突变体将有助于TNFR的功能分析。通过聚合酶链反应对受体结合界面中的六个氨基酸（靠近TNF残基30、80和140）进行随机突变。构建了两个包含超过500万个TNF突变体的噬菌体文库。通过筛选对TNFR1或TNFR2无亲和力的突变体，我们分别成功分离出4个TNFR2选择性候选物和16个TNFR1选择性候选物。TNFR1选择性候选物在残基30附近高度突变，而TNFR2选择性候选物在残基140附近高度突变，尽管两者在残基140和30附近分别具有保守序列。这一发现表明噬菌体展示技术适用于鉴定TNF与TNFR1和TNFR2相互作用的重要区域。纯化的克隆R1-6是一种TNFR1选择性候选物，保持完全生物活性，对TNFR1具有完全亲和力且不激活TNFR2，表明R1-6 TNF突变体在分析TNFR1受体功能方面的有用性。为了进一步阐明R1-6的受体选择性，我们通过X射线晶体学检查了R1-6的结构。结果表明，R31A和R32G突变强烈影响与TNFR2的静电相互作用，而L29K突变有助于R1-6与TNFR1的结合。这种噬菌体展示技术可用于高效构建功能突变体以分析TNF的结构-功能关系，这可能有助于基于受体选择性的计算机辅助药物设计。
