Unraveling the mechanism of the lactose permease of Escherichia coli

Abstract

We studied the effect of pH on ligand binding in wild-type lactose permease or mutants in the four residues-Glu-269, Arg-302, His-322, and Glu-325-that are the key participants in H(+) translocation and coupling between sugar and H(+) translocation. Although wild-type permease or mutants in Glu-325 and Arg-302 exhibit marked decreases in affinity at alkaline pH, mutants in either His-322 or Glu-269 do not titrate. The results offer a mechanistic model for lactose/H(+) symport. In the ground state, the permease is protonated, the H(+) is shared between His-322 and Glu-269, Glu-325 is charge-paired with Arg-302, and substrate is bound with high affinity at the outside surface. Substrate binding induces a conformational change that leads to transfer of the H(+) from His-322/Glu-269 to Glu-325 and reorientation of the binding site to the inner surface with a decrease in affinity. Glu-325 then is deprotonated on the inside because of rejuxtaposition with Arg-302. The His-322/Glu-269 complex then is reprotonated from the outside surface to reinitiate the cycle.

Figure 1

Model for proposed ground-state conformation of lac permease, which binds substrate with high affinity. For clarity, 6 of the 12 helices in the permease are shown. Glu-126 (helix IV) and Arg-144 (helix V) are charge-paired and with Cys-148 (helix V) comprise the major components of the substrate binding site. Arg-302 (helix IX) is charge-paired with Glu-325 (helix X), while protonated His-322 (helix X) interacts with Glu-269 (helix VIII). Thus, the relevant H⁺ is shared by His-322 and Glu-269. Also shown are the charge pair between Asp-240 (helix VII) and Lys-319 (helix X), which are not essential for the mechanism. See text for further details regarding the transport mechanism.

Figure 2

Effect of TDG on NEM labeling of Cys-148 in single-Cys-148 permease or mutants E325Q/Cys-148, E325D/Cys-148, R302A/Cys-148, R302K/Cys-148, H322A/Cys-148, H322N/Cys-148, H322Q/Cys-148, and E269D/Cys-148 at pH 5.5, pH 7.5, and pH 9.5. (Left) Right-side-out membrane vesicles were incubated with 0.5 mM (at pH 5.5 and 7.5) or 0.22 mM (at pH 9.5) [¹⁴C]NEM at 25°C, for 15 min (pH 5.5), 5 min (pH 7.5), or 2 min (pH 9.5) in the absence or presence of given concentrations of TDG. Reactions were quenched with DTT, and biotinylated permease was solubilized and purified by affinity chromatography on monomeric avidin. Aliquots of protein were separated on a SDS/12% polyacrylamide gel, and [¹⁴C]-labeled permease was visualized by autoradiography. Although not shown, a fraction of the protein was analyzed by Western blotting to determine the amount of permease in each sample; no significant differences were observed. (Right) Incorporation of [¹⁴C]NEM was quantitated by a Storm 860 PhosphorImager, and labeling in the presence of various concentrations of TDG is expressed as % labeling observed in the absence of TDG. ○, pH 5.5; ■, pH 7.5; ●, pH 9.5.