Reed C, Fu Z Q, Wu J, Xue Y N, Harrison R W, Chen M J, Weber I T
Department of Microbiology and Immunology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA 19107, USA.
Protein Eng. 1997 Oct;10(10):1101-7. doi: 10.1093/protein/10.10.1101.
Crystal structures have been determined of recombinant human tumor necrosis factor-alpha (TNF-alpha) and its R31D mutant that preferentially binds to TNF receptor R1 with more than seven times the relative affinity of binding to receptor R2. Crystals of the wild-type TNF were of space group P4(1)2(1)2 and had unit cell dimensions of a = b = 94.7 and c = 117.4 A. Refinement of the structure gave an R-factor of 22.3% at 2.5 A resolution. The crystals of TNF R31D mutant diffracted to 2.3 A resolution, and were of identical space group to the wild type with unit cell dimensions of a = b = 95.4 and c = 116.2 A, and the structure was refined to an R-factor of 21.8%. The trimer structures of the wild-type and mutant TNF were similar with a root mean square (r.m.s.) deviation of 0.56 A for Calpha atoms; however, the subunits within each trimer were more variable with an average r.m.s. deviation of 1.00 A on Calpha atoms for pairwise comparison of subunits. Model complexes of TNF with receptors R1 and R2 have been used to predict TNF-receptor interactions. Arg31 in all three subunits of wild-type TNF is predicted to form an ionic interaction with the equivalent glutamic acid in both receptors R1 and R2. Asp31 of the TNF R31D mutant is predicted to interact differently with the two receptors. The side chain of Asp31 in two subunits of the TNF mutant is predicted to form hydrogen bond interactions with Ser59 or Cys70 of R1, while it has no predicted interactions with R2. The loss of three strong ionic interactions of Arg31 and the electrostatic repulsion of Asp31 with Glu in the receptors is consistent with the reduced binding of the R31D mutant to both receptors relative to wild-type TNF. The replacement of these ionic interactions by two weaker hydrogen bond interactions between Asp31 of the R31D mutant and R1, compared with no interactions with R2, is in agreement with the observed preferential binding of the R31D mutant to R1 over R2. Analysis of the structure and function of receptor-discriminating mutants of TNF will help understand the biological role of TNF and facilitate its use as an antitumor agent.
已确定重组人肿瘤坏死因子-α（TNF-α）及其R31D突变体的晶体结构，该突变体优先结合TNF受体R1，其与受体R2结合的相对亲和力超过7倍。野生型TNF的晶体属于空间群P4(1)2(1)2，晶胞参数为a = b = 94.7 Å，c = 117.4 Å。在2.5 Å分辨率下对结构进行精修，得到的R因子为22.3%。TNF R31D突变体的晶体衍射到2.3 Å分辨率，与野生型属于相同的空间群，晶胞参数为a = b = 95.4 Å，c = 116.2 Å，结构精修后的R因子为21.8%。野生型和突变型TNF的三聚体结构相似，α碳原子的均方根（r.m.s.）偏差为0.56 Å；然而，每个三聚体内的亚基差异更大，亚基两两比较时α碳原子的平均r.m.s.偏差为1.00 Å。TNF与受体R1和R2的模型复合物已用于预测TNF-受体相互作用。预测野生型TNF所有三个亚基中的精氨酸31会与受体R1和R2中的等效谷氨酸形成离子相互作用。预计TNF R31D突变体的天冬氨酸31与两种受体的相互作用不同。预计TNF突变体两个亚基中天冬氨酸31的侧链会与R1的丝氨酸59或半胱氨酸70形成氢键相互作用，而预计其与R2没有相互作用。精氨酸31的三个强离子相互作用的丧失以及天冬氨酸31与受体中谷氨酸的静电排斥与R31D突变体相对于野生型TNF与两种受体结合减少一致。与R2无相互作用相比，R31D突变体的天冬氨酸31与R1之间通过两个较弱的氢键相互作用取代这些离子相互作用，这与观察到的R31D突变体对R1的优先结合优于R2一致。对TNF受体区分突变体的结构和功能分析将有助于理解TNF的生物学作用，并促进其作为抗肿瘤药物的应用。
