Goh C R, Loh C S, Porter A G
Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge Crescent.
Protein Eng. 1991 Oct;4(7):785-91. doi: 10.1093/protein/4.7.785.
Single amino acid substitutions were generated in predicted hydrophilic loop regions of the human tumour necrosis factor beta (TNF-beta) molecule, and the mutant proteins were expressed in Escherichia coli and purified. Mutants with single amino acid changes at either of two distinct loop regions, at positions aspartic acid 50 or tyrosine 108, were found to have greatly reduced receptor binding and cytotoxic activity. These two regions in TNF-beta correspond to known loop regions where mutations also result in loss of biological activity of TNF-alpha, a related cytokine which shares the same cellular receptors with TNF-beta. The two distinct loops at positions 31-34 and 84-89 in the known three-dimensional structure of TNF-alpha (equivalent to positions 46-50 and 105-110 respectively in TNF-beta), lie on opposite sides of the TNF-alpha monomer. When the TNF-alpha monomer forms a trimer, the two loops, each from a different subunit of the trimer, come together and lie in a cleft between adjacent subunits. Together, these findings suggest that a TNF receptor binds to a cleft between subunits via surface loops at amino acid residues 31-34 and 84-89 in TNF-alpha, and similarly via surface loops including amino acids aspartic acid 50 and tyrosine 108 in TNF-beta.
在人类肿瘤坏死因子β（TNF-β）分子预测的亲水性环区域产生了单氨基酸取代，并在大肠杆菌中表达和纯化了突变蛋白。发现在两个不同环区域之一（天冬氨酸50位或酪氨酸108位）发生单氨基酸变化的突变体，其受体结合和细胞毒性活性大大降低。TNF-β中的这两个区域对应于已知的环区域，在这些区域突变也会导致TNF-α（一种与TNF-β共享相同细胞受体的相关细胞因子）丧失生物活性。在TNF-α已知的三维结构中，31-34位和84-89位的两个不同环（分别相当于TNF-β中的46-50位和105-110位）位于TNF-α单体的相对两侧。当TNF-α单体形成三聚体时，这两个环（每个环来自三聚体的不同亚基）聚集在一起并位于相邻亚基之间的裂隙中。这些发现共同表明，TNF受体通过TNF-α中31-34位和84-89位氨基酸残基处的表面环与亚基之间的裂隙结合，并且类似地通过包括TNF-β中天冬氨酸50和酪氨酸108等氨基酸的表面环结合。
