USE OF DESIGNED METAL-BINDING SITES TO STUDY HELIX PROXIMITY IN THE LACTOSE PERMEASE OF ESCHERICHIA COLI .2. PROXIMITY OF HELIX IX (ARG302) WITH HELIX X (HIS322 AND GLU325)

摘要

Engineering divalent metal-binding sites into the lactose permease of Escherichia coli by introducing bis-His residues has been utilized to confirm the proximity of helices VIII (Glu269-->His) and X (His322) [Jung, K., Voss, J., He, M., Hubbell, W. L., & Kaback, H. R. (1995) Biochemistry 34, 6272] and helices VII (Asp237-->His) and XI (Lys358-->His) [He, M. M., Voss, J., Hubbell, W. L., & Kaback, H. R. (1995) Biochemistry 34, 00000-00000]. In this paper, the approach is used to confirm and extend the relationship between helices IX (Arg302) and X (His322 and Glu325) [Jung, K., Jung, H., Wu, J., Prive, G. G., & Kaback, H. R. (1993) Biochemistry 32, 12273]. Thus, mutants Arg30-->His, Glu325-->His, and Arg302-His/Glu325-->His were constructed, and Mn2+ binding was assayed by electron paramagnetic resonance. Mutant Arg302-->His binds Mn2+ with a K-D of about 24 mu M and a stoichiometry approximating unity in all likelihood because the His residue at position 302 forms a metal-binding site in conjunction with the native His residue at position 322. Mutant Arg302-->His/Glu325-->His also binds Mn2+ with a 1:1 stoichiometry, but the K-D is decreased to about 13 mu M. The results suggest that Arg302 is sufficiently close to both Glu325 and His322 to form a tridentate metal-binding site in mutant Arg302-->His/Glu325-->His. In contrast, replacement of Glu325 with His in permease with a native His residue at position 322 does not lead to Mn2+ binding, The results provide strong support for the helix packing model proposed.