Engineering conformational flexibility in the lactose permease of Escherichia coli: Use of glycine-scanning mutagenesis to rescue mutant Glu325 -> Asp

Lactose/H+ symport by lactose permease of Escherichia coli involves interac tions between four irreplaceable charged residues in transmembrane helices that play essential roles in Hf translocation and coupling [Glu269 (helix V III) with His322 (helix X) and Arg302 (helix IX) with Glu325 (helix X)], as well as Glu126 (helix IV) and Arg144 (helix V) which are obligatory for su bstrate binding. The conservative mutation Glu325 --> Asp causes a 10-fold reduction in the V-max for active lactose transport and markedly decreased lactose-induced H+ influx with no effect on exchange or counterflow, neithe r of which involves H+ symport. Thus, shortening the side chain may weaken the interaction of the carboxyl group at position 325 with the guanidino gr oup of; Arg302. Therefore, Gly-scanning mutagenesis of helices IX and X and the intervening loop was employed systematically with mutant Glu325 --> As p in an effort to rescue function by introducing conformational flexibility between the two helices. Five Gly replacement mutants in the Glu325 --> As p background are identified that exhibit significantly higher transport act ivity. Furthermore, mutant Val316 --> Gly/Glu325 --> Asp catalyzes active t ransport, efflux, and lactose-induced H+ influx with kinetic properties app roaching those of wild-type permease. It is proposed that introduction of c onformational flexibility at the interface between helices IX and X improve s juxtapositioning between Arg302 and Asp325 during turnover, thereby allow ing more effective deprotonation of the permease on the inner surface of th e membrane [Sahin-Toth, M., Karlin, A., and Kaback, H. R. (2000) Proc. Natl . Acad. Sci. U.S.A. 97, 10729-10732.