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)

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 Arg302→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/μM 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 μM. 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.