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  • Title: 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.
    Author: He MM, Voss J, Hubbell WL, Kaback HR.
    Journal: Biochemistry; 1997 Nov 04; 36(44):13682-7. PubMed ID: 9354638.
    Abstract:
    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.
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