Arginine 302 (helix IX) in the lactose permease of Escherichia coli is in close proximity to glutamate 269 (helix VIII) as well as glutamate 325.
By using a variety of biochemical and biophysical approaches, a helix packing model for the lactose permease of Escherichia coli has been proposed in which the four residues that are irreplaceable with respect to coupling are paired--Glu269 (helix VIII) with His322 (helix X) and Arg302 (helix XI) with Glu325 (helix X). In addition, the substrate translocation pathway is located at the interface between helices V and VIII, which is in close vicinity to the four essential residues. Based on this structural information and functional studies of mutants in the four irreplaceable residues, a molecular mechanism for energy coupling in the permease has been proposed [Kaback, H. R. (1997) Pro.c Natl. Acad. Sci. U.S.A. 94, 5539]. The principle idea of this model is that Arg302 interacts with either Glu325 or Glu269 during turnover. Evidence that Arg302 is in close proximity with Glu325 has been presented [Jung, K., Jung, H., Wu, J., Prive, G. G., & Kaback, H. R. (1993) Biochemistry 32, 12273; He, M. M., Voss, J., Hubbell, W. L., & Kaback, H. R. (1995) Biochemistry 34, 15667]; however, the proximity of Arg302 to Glu269 has not been examined. In this report, it is shown by two methods that Arg302 is also close to Glu269: (i) permease with Glu269-->His, Arg302-->His, and His322-->Phe binds Mn2+ with high affinity at pH 7.5, but not at pH 5.5; and (ii) site-directed spin-labeling of the double Cys mutant Glu269-->Cys/Arg302-->Cys exhibits spin-spin interaction with an interspin distance of about 14-16 A. In addition, the spin-spin interaction is stronger and interspin distance shorter after the permease is reconstituted into proteoliposomes. Taken as a whole, the data are consistent with the idea that Arg302 may interact with either Glu325 or Glu269 during turnover.
He
M M
MM
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095-1662, USA.
Voss
J
J
Hubbell
W L
WL
Kaback
H R
HR
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005615:Freezing; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D008345:Manganese; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D013113:Spin Labels; D027981:Symporters; D013696:Temperature
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013113:Spin Labels; D027981:Symporters; D018698:Glutamic Acid; D008345:Manganese; C021499:lactose permease; D001120:Arginine