Gokce I, Raggett E M, Hong Q, Virden R, Cooper A, Lakey J H
Department of Chemistry Faculty of Science, Gaziomanpasa University, Tokat, Turkey.
J Mol Biol. 2000 Dec 8;304(4):621-32. doi: 10.1006/jmbi.2000.4232.
Colicins translocate across the Escherichia coli outer membrane and periplasm by interacting with several receptors. After first binding to the outer membrane surface receptors via their central region, they interact with TolA or TonB proteins via their N-terminal region. Colicin N residues critical to TolA binding have been discovered, but the full extent of any colicin TolA site is unknown. We present, for the first time, a fully mapped TolA binding site for a colicin. It was determined through the use of alanine-scanning mutants, glutathione S-transferase fusion peptides and Biacore/fluorescence binding studies. The minimal TolA binding region is 27 residues and of similar size to the TolA binding region of bacteriophage g3p-D1 protein. Stopped-flow kinetic studies show that the binding to TolA follows slow association kinetics. The role of other E. coli Tol proteins in colicin translocation was also investigated. Isothermal titration microcalorimetry (ITC) and in vivo studies conclusively show that colicin N translocation does not require the presence of TolB. ITC also demonstrated colicin A interaction with TolB, and that colicin A in its native state does not interact with TolAII-III. Colicin N does not bind TolR-II. The TolA protein is shown to be unsuitable for direct immobilisation in Biacore analysis.
大肠杆菌素通过与多种受体相互作用穿过大肠杆菌外膜和周质。它们首先通过中央区域与外膜表面受体结合，然后通过N端区域与TolA或TonB蛋白相互作用。已经发现了对TolA结合至关重要的大肠杆菌素N残基，但任何大肠杆菌素TolA位点的完整范围尚不清楚。我们首次展示了一种大肠杆菌素的完整映射TolA结合位点。它是通过使用丙氨酸扫描突变体、谷胱甘肽S-转移酶融合肽以及Biacore/荧光结合研究确定的。最小的TolA结合区域为27个残基，大小与噬菌体g3p-D1蛋白的TolA结合区域相似。停流动力学研究表明，与TolA的结合遵循缓慢的缔合动力学。还研究了其他大肠杆菌Tol蛋白在大肠杆菌素转运中的作用。等温滴定量热法（ITC）和体内研究确凿地表明，大肠杆菌素N的转运不需要TolB的存在。ITC还证明了大肠杆菌素A与TolB的相互作用，并且天然状态的大肠杆菌素A不与TolAII-III相互作用。大肠杆菌素N不结合TolR-II。结果表明，TolA蛋白不适合在Biacore分析中直接固定。
