Proximity of periplasmic loops in the lactose permease of Escherichia coli determined by site-directed cross-linking.
Out of over 60 single-Cys mutants in putative periplasmic loops in lactose permease, three mutants [Tyr101 --> Cys (loop III/IV), Leu313 --> Cys (loop IX/X), and Ser375 --> Cys (loop XI/XII)] spontaneously form disulfide-linked dimers, indicating that these loops are located on the periphery of the 12-helix bundle that comprises the permease. By using a permease construct with a factor Xa protease site in the middle cytoplasmic loop, cross-linking between paired-Cys residues in the N- and C-terminal halves of the permease was studied by spontaneous or copper-(1, 10-phenanthroline)3-catalyzed disulfide formation or by cross-linking with homo- or heterobifunctional reagents in which the distance between the reactive groups and the flexibility of the linker vary. The findings suggest that the longer loops are relatively flexible; however, cross-linking of residues between loops is specific, indicating that these domains are not simply flexible, hydrophilic connections between helices that interact randomly. More specifically, the findings indicate that the first periplasmic loop (loop I/II) is close to loops VII/VIII and XI/XII, placing helix XII in close proximity to helices II and XI. In addition, the observations are consistent with previous results [Wu, J., & Kaback, H. R. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 14498-502] demonstrating that helices I and II are close to helices VII and XI. Finally, evidence is presented indicating that conformational flexibility between loops I/II and XI/XII may be important for permease turnover.
Sun
J
J
Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095-1662, USA.
Kaback
H R
HR
D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D015951:Factor Xa / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D027981:Symporters
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D015951:Factor Xa; D003545:Cysteine