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 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 Arg144 (helix V) which are obligatory for substrate 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, 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 Val316-->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-Tóth, M., Karlin, A., and Kaback, H. R. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 10729-10732.
Weinglass
A B
AB
Howard Hughes Medical Institute, Department of Physiology and of Microbiology & Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095-1662, USA.
Smirnova
I N
IN
Kaback
H R
HR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics*; D001693:Biological Transport, Active / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000235:genetics*; D005998:Glycine / Q000235:genetics*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000235:genetics; D027981:Symporters; D014633:Valine / Q000235:genetics
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D001224:Aspartic Acid; D018698:Glutamic Acid; C021499:lactose permease; D014633:Valine; D005998:Glycine