Use of designed metal-binding sites to study helix proximity in the lactose permease of Escherichia coli. 1. Proximity of helix VII (Asp237 and Asp240) with helices X (Lys319) and XI (Lys358)

The lactose permease of Escherichia coli contains two pairs of oppositely charged residues that interact functionally, Asp240 (helix VII)/Lys319 (helix X) and Asp237 (helix VII)/Lys358 (helix XI). Single- and double-His replacement mutants at these positions have been constructed and characterized with respect to transport activity and Mn²⁺ binding. The following results confirm the functional interactions between both sets of residues: (i) At pH 7.5, where the imidazole is likely to be unprotonated, the double-His mutants Asp237→His/Lys358→His and Asp240→His/Lys319→His exhibit significant transport activity while the single-His mutants Lys319→His and Lys358-His are inactive, (ii) At pH 5.5, where the imidazole is likely to be protonated, the double-His mutants Asp240→His/Lys319→His and Asp237→His/ Lys358→→His are inactive; however, the single-His mutant Lys319→His exhibits significant activity, (iii) The single-His mutant Asp237→His or Asp240→His is inactive at all pH values tested. In addition, a pH titration of Asp237→His/Lys→His permease activity exhibits a midpoint at about 6.2. Finally, the purified mutant proteins Asp237→His/Lys358→His and Asp240→His/Lys319→His were assayed for Mn²⁺ binding by electron paramagnetic resonance spectroscopy. Asp237→His/Lys358→His permease binds Mn²⁺ with a stoichiometry of unity at pH 7.5, but much less binding is observed at pH 5.5, demonstrating directly that helix VII (Asp237) is in close proximity to helix XI (Lys358). In contrast, Asp240→His/Lys319→His permease does not bind Mn²⁺, indicating that these two residues interact over a longer distance. In the following paper, the same approach is used to confirm the close proximity of helices IX (Arg302) and X (His322 and Glu325).