Engineering Conformational Flexibility in the Lactose Permease of Escherichia coli: Use of Glycine-Scanning Mutagenesis To Rescue Mutant Glu325 -> Asp

摘要

Lactose/H+ symport by lactose permease of Escherichia coli involves interactions between four irreplaceable charged residues in transmembrane helices that play essential roles in H+ translocation and coupling [Glu269 (helix VIII) with His322 (helix X) and Arg302 (helix IX) with Glu325 (helix X)], as well as Glu126 (helix IV) and Argl44 (helix V) which are obligatory for substrate binding. The conservative mutation Glu325-Asp causes a lO-fold reduction in the V max for active lactose transport and markedly decreased lactose-induced H+ influx with no effect on exchange or counterflow, neither of which involves H+ symport. Thus, shortening the side chain may weaken the interaction of the carboxyl group at position 325 with the guanidino group of Arg302. Therefore, Gly-scanning mutagenesis of helices IX and X and the intervening loop was employed systematically with mutant Glu325-Asp in an effort to rescue function by introducing conformational flexibility between the two helices. Five Gly replacement mutants in the Glu325-Asp background are identified that exhibit significantly higher transport activity. Furthermore, mutant Va13l6-Gly/Glu325-Asp catalyzes active transport, efflux, and lactose-induced H+ influx with kinetic properties approaching those of wild-type permease. It is proposed that introduction of conformational flexibility at the interface between helices IX and X improves juxtapositioning between Arg302 and Asp325 during turnover, thereby allowing more effective deprotonation of the permease on the inner surface of the membrane [Sahin- T6th, M., Karlin, A., and Kaback, H. R. (2000) Proc. Natl. A(;qd, S(;i, U,S,A, 97. 10729-10732.