Suppressor scanning at positions 177 and 236 in the Escherichia coli lactose/H+ cotransporter and stereotypical effects of acidic substituents that suggest a favored orientation of transmembrane segments relative to the lipid bilayer

Abstract

Acidic substituents for Ala-177 (helix 6) or Tyr-236 (helix 7) in LacY cause effects on sugar recognition and cosubstrate coupling that are stereotypical of neutral substituents. Thus, helices 6 and 7 are probably oriented to produce little side-chain contact with the low dielectric lipid bilayer at positions 177 and 236.

FIG. 1

Growth-inhibiting toxicity of LacY mutants that carry acidic substitutions at positions 177 and 236. Optical density was monitored in cultures with (open symbols) or without (solid symbols) 1 mM IPTG to induce plasmid-borne lacY expression. (A) Growth of cells with wild-type LacY (circles) or A177D (squares) or A177E (triangles) mutant LacY. (B) Growth of cells with wild-type LacY (circles) or Y236D (squares) or Y236E (triangles) mutant LacY.

FIG. 2

Effect of TDG on the transmembrane ΔpH. For the indicated LacY mutants, the transmembrane ΔpH was monitored either in the presence or in the absence (control) of 1 mM TDG.

FIG. 3

Retention of high TDG affinity by LacY mutants carrying acidic substitutions at positions 177 and 236. The transmembrane ΔpH was monitored as a function of TDG concentration in E. coli expressing either wild-type LacY (•) or the A177E (▵), A177D (▴), Y236D (□), or Y236E (▪) LacY mutant.

FIG. 4

A working model of the LacY tertiary structure. The solid ovals emphasize the positions of helix 6 (Ala-177) and helix 7 (Tyr-236) within the context of Brooker’s symmetrical lac permease model (4, 14). These helices are probably oriented so as to shield positions 177 and 236 from intimate contact with the lipid environment.