Vinothkumar Kutti R, McMullan Greg, Henderson Richard
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
Structure. 2014 Apr 8;22(4):621-7. doi: 10.1016/j.str.2014.01.011. Epub 2014 Mar 6.
Binding of a single-chain Fv antibody to Escherichia coli β-galactosidase (β-gal) is known to stabilize the enzyme and activate several inactive point mutants, historically called antibody-mediated enzyme formation mutants. To understand the nature of this activation, we have determined by electron cryo-microscopy the structure of the complex between β-gal and the antibody scFv13R4. Our structure localizes the scFv13R4 binding site to the crevice between domains 1 and 3 in each β-gal subunit. The mutations that scFv13R4 counteracts are located between the antibody binding site and the active site of β-gal, at one end of the TIM-barrel that forms domain 3 where the substrate lactose is hydrolyzed. The mode of binding suggests how scFv stabilizes both the active site of β-gal and the tetrameric state.
已知单链Fv抗体与大肠杆菌β-半乳糖苷酶（β-gal）结合可稳定该酶，并激活几个无活性的点突变体，这些突变体在历史上被称为抗体介导的酶形成突变体。为了了解这种激活的本质，我们通过电子冷冻显微镜确定了β-gal与抗体scFv13R4之间复合物的结构。我们的结构将scFv13R4结合位点定位在每个β-gal亚基的结构域1和结构域3之间的缝隙处。scFv13R4能抵消的突变位于β-gal的抗体结合位点和活性位点之间，在形成结构域3的TIM桶的一端，底物乳糖在此处被水解。结合模式表明了scFv如何稳定β-gal的活性位点和四聚体状态。
