Baxa U, Steinbacher S, Miller S, Weintraub A, Huber R, Seckler R
Universtät Regensburg, Institut für Biophysik und Physikalische Biochemie, Germany.
Biophys J. 1996 Oct;71(4):2040-8. doi: 10.1016/S0006-3495(96)79402-X.
Bacteriophage P22 binds to its cell surface receptor, the repetitive O-antigen structure in Salmonella lipopolysaccharide, by its six homotrimeric tailspikes. Receptor binding by soluble tailspikes and the receptor-inactivating endorhamnosidase activity of the tailspike protein were studied using octa- and dodecasaccharides comprising two and three O-antigen repeats of Salmonella enteritidis and Salmonella typhimurium lipopolysaccharides. Wild-type tailspike protein and three mutants (D392N, D395N, and E359Q) with defective endorhamnosidase activity were used. Oligosaccharide binding to all three subunits, measured by a tryptophan fluorescence quench or by fluorescence depolarization of a coumarin label attached to the reducing end of the dodecasaccharide, occurs independently. At 10 degrees C, the binding affinities of all four proteins to oligosaccharides from both bacterial strains are identical within experimental error, and the binding constants for octa- and dodecasaccharides are 1 x 10(6) M(-1) and 2 x 10(6) M(-1), proving that two O-antigen repeats are sufficient for lipopolysaccharide recognition by the tailspike. Equilibration with the oligosaccharides occurs rapidly, but the endorhamnosidase produces only one cleavage every 100 s at 10 degrees C or about 2 min(-1) at the bacterial growth temperature. Thus, movement of virions in the lipopolysaccharide layer before DNA injection may involve the release and rebinding of individual tailspikes rather than hydrolysis of the O-antigen.
噬菌体P22通过其六个同三聚体尾钉蛋白与细胞表面受体——沙门氏菌脂多糖中的重复O抗原结构结合。使用包含肠炎沙门氏菌和鼠伤寒沙门氏菌脂多糖的两个和三个O抗原重复序列的八糖和十二糖，研究了可溶性尾钉蛋白与受体的结合以及尾钉蛋白的受体失活内鼠李糖苷酶活性。使用了野生型尾钉蛋白和三种具有缺陷内鼠李糖苷酶活性的突变体（D392N、D395N和E359Q）。通过色氨酸荧光猝灭或附着在十二糖还原端的香豆素标签的荧光去极化测量的寡糖与所有三个亚基的结合是独立发生的。在10℃下，在实验误差范围内，所有四种蛋白质与两种细菌菌株的寡糖的结合亲和力相同，八糖和十二糖的结合常数分别为1×10⁶ M⁻¹和2×10⁶ M⁻¹，证明两个O抗原重复序列足以被尾钉蛋白识别脂多糖。与寡糖的平衡迅速发生，但内鼠李糖苷酶在10℃下每100秒仅产生一次切割，在细菌生长温度下约为2分钟⁻¹。因此，在DNA注射前病毒粒子在脂多糖层中的移动可能涉及单个尾钉蛋白的释放和重新结合，而不是O抗原的水解。
