Sun J, Li J, Carrasco N, Kaback H R
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095-1662, USA.
Biochemistry. 1997 Jan 7;36(1):274-80. doi: 10.1021/bi962292f.
Monoclonal antibody (mAb) 4B11 binds to a conformational epitope in the lactose permease that is exposed on the cytoplasmic face of the membrane with a KD of 2.8 x 10(-7) M. By studying binding of 4B11 to permease mutants containing six contiguous His residues in each cytoplasmic loop, inserted factor Xa protease sites, or a C-terminal deletion, the cytoplasmic loops between helices VIII and IX (loop VIII/IX) and between helices X and XI (loop X/XI) are shown to comprise the epitope. Subsequently, Cys-scanning mutagenesis in conjunction with thiol modification was carried out in order to identify specific residues involved in 4B11 recognition. Glu342 and Arg344 in loop X/XI are primary determinants for 4B11 binding, while Ile283 in loop VIII/IX and Phe334 and Lys335 in loop X/XI are secondary determinants. Consistently, binding of avidin to biotinylated single-Cys replacements in loop VIII/IX or loop X/XI blocks 4B11 binding, but avidin binding to biotinylated Cys residues in other cytoplasmic loops or insertion of cytochrome b562 into cytoplasmic loop VI/VII has no significant effect. The studies demonstrate that the last two cytoplasmic loops in lactose permease comprise a discontinuous epitope for monoclonal antibody 4B11 and thereby provide independent evidence for the conclusion that helices VIII-XI are in close proximity.
单克隆抗体（mAb）4B11与乳糖通透酶上的一个构象表位结合，该表位暴露于膜的胞质面，解离常数KD为2.8×10⁻⁷M。通过研究4B11与每个胞质环中含有六个连续组氨酸残基、插入的因子Xa蛋白酶切位点或C末端缺失的通透酶突变体的结合情况，发现螺旋VIII和IX之间的胞质环（环VIII/IX）以及螺旋X和XI之间的胞质环（环X/XI）构成了该表位。随后，进行了半胱氨酸扫描诱变并结合硫醇修饰，以鉴定参与4B11识别的特定残基。环X/XI中的Glu342和Arg344是4B11结合的主要决定因素，而环VIII/IX中的Ile283以及环X/XI中的Phe334和Lys335是次要决定因素。同样，抗生物素蛋白与环VIII/IX或环X/XI中生物素化的单半胱氨酸替代物的结合会阻断4B11的结合，但抗生物素蛋白与其他胞质环中生物素化的半胱氨酸残基的结合或细胞色素b562插入胞质环VI/VII没有显著影响。这些研究表明，乳糖通透酶的最后两个胞质环构成了单克隆抗体4B11的一个不连续表位，从而为螺旋VIII - XI紧密相邻的结论提供了独立证据。
