BINDING OF LIGAND OR MONOCLONAL-ANTIBODY 4B1 INDUCES DISCRETE STRUCTURAL-CHANGES IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

By using Cys-scanning mutagenesis with site-directed sulfhydryl modifi cation in situ [Frillingos, S., & Kaback, H. R. (1996) Biochemistry 35 , 3950-3956], conformational changes induced by binding of ligand or m onoclonal antibody (mAb) 4B1 in the lactose permease of Escherichia co li 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 vesic les. In addition, site-directed fluorescence spectroscopy shows that m Ab 4B1 binding causes position 331 (helix X) in the permease to experi ence a more hydrophobic environment. In contrast, ligand binding elici ts more widespread changes, as evidenced by enhancement of the NEM rea ctivity of Ala244-->Cys, Thr248-->Cys (helix VII), Thr265-->Cys (helix VIII), Val315-->Cys (helix X), Gln359-->Cys, or Met362-->Cys (helix X I) permease, none of which are altered by 4B1 binding. Furthermore, no effect of 4B1 is observed on the reactivity of Cys148 (helix V), Val2 64-->Cys, Gly268-->Cys, or Asn272-->Cys (helix VIII), positions which probably make direct contact with substrate. With respect to the N-ter minal half of the permease, 4B1 binding causes a small increase in the reactivity of mutants Pro28-->Cys or Pro31-->Cys (helix I), while lig and 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.