Binding of ligand or monoclonal antibody 4B1 induces discrete structural changes in the lactose permease of Escherichia coli
Binding of ligand or monoclonal antibody 4B1 induces discrete structural changes in the lactose permease of Escherichia coli
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DOI：
10.1021/bi970233b
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发表时间：
1997-05-27
期刊：
影响因子：
2.9
通讯作者：
Kaback, HR
中科院分区：
文献类型：
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作者：
Frillingos, S;Wu, JH;Kaback, HR
采用半胱氨酸扫描原位巯基修饰[Frillingos，S.，&Kaback，H.R.(1996)BioChemical 35,3950-3956]的方法，研究了配体或单抗4B1与大肠杆菌乳糖渗透酶结合后的构象变化。在螺旋I、V、VII、、X或XI的31个单半胱氨酸替换突变体中，4B1结合改变了右侧膜泡中Val238-->；半胱氨酸(螺旋VII)、Val331-->；半胱氨酸(螺旋X)或单半胱氨酸355(螺旋XI)渗透酶与N-乙基马来酰亚胺(NEM)的反应活性。此外，定点荧光光谱显示，mAb4B1结合导致渗透酶中的位置331(螺旋X)经历更疏水的环境。相反，配体结合引起更广泛的变化，如Ala244-->；Cys，Thr248-->；Cys(螺旋VII)，Thr265-->；Cys(螺旋VIII)，Val315-->；Cys(螺旋X)，Gln359-->；Cys，或Met362-->；Cys(螺旋XI)渗透酶的NEM反应活性增强，这些都不被4B1结合改变。此外，4B1对Cys148(螺旋V)、Val264-->；Cys、Gly268-->；Cys或Asn272-->；Cys(螺旋VIII)的反应性没有影响，这些位置可能与底物直接接触。对于渗透酶的N-末端一半，4B1结合导致突变体Pro28->；Cys或Pro31->；Cys(螺旋I)的反应性略有增加，而配体结合引起更大的反应性增加。结果表明，4B1结合引起的通透酶结构变化比配体结合引起的结构变化要小得多。
By using Cys-scanning mutagenesis with site-directed sulfhydryl modification in situ [Frillingos, S., & Kaback, H. R. (1996) Biochemistry 35, 3950-3956], conformational changes induced by binding of ligand or monoclonal antibody (mAb) 4B1 in the lactose permease of Escherichia coli were studied. Out of 31 single-Cys replacement mutants in helices I, V, VII, VIII, X, or XI, 4B1 binding alters the reactivity of Val238-->Cys (helix VII), Val331-->Cys (helix X), or single-Cys355 (helix XI) permease with N-ethylmaleimide (NEM) in right-side-out membrane vesicles. In addition, site-directed fluorescence spectroscopy shows that mAb 4B1 binding causes position 331 (helix X) in the permease to experience a more hydrophobic environment. In contrast, ligand binding elicits more widespread changes, as evidenced by enhancement of the NEM reactivity of Ala244-->Cys, Thr248-->Cys (helix VII), Thr265-->Cys (helix VIII), Val315-->Cys (helix X), Gln359-->Cys, or Met362-->Cys (helix XI) permease, none of which are altered by 4B1 binding. Furthermore, no effect of 4B1 is observed on the reactivity of Cys148 (helix V), Val264-->Cys, Gly268-->Cys, or Asn272-->Cys (helix VIII), positions which probably make direct contact with substrate. With respect to the N-terminal half of the permease, 4B1 binding causes a small increase in the reactivity of mutants Pro28-->Cys or Pro31-->Cys (helix I), while ligand binding causes much greater increases in reactivity. The findings indicate that 4B1 binding induces a structural change in the permease that is much less widespread than that induced by ligand binding.