Sugar binding induces the same global conformational change in purified LacY as in the native bacterial membrane

Abstract

Many independent lines of evidence indicate that the lactose permease of Escherichia coli (LacY) is highly dynamic and that sugar binding causes closing of a large inward-facing cavity with opening of a wide outward-facing hydrophilic cavity. Therefore, lactose/H(+) symport catalyzed by LacY very likely involves a global conformational change that allows alternating access of single sugar- and H(+)-binding sites to either side of the membrane (the alternating access model). The x-ray crystal structures of LacY, as well as the majority of spectroscopic studies, use purified protein in detergent micelles. By using site-directed alkylation, we now demonstrate that sugar binding induces virtually the same global conformational change in LacY whether the protein is in the native bacterial membrane or is solubilized and purified in detergent. The results also indicate that the x-ray crystal structure reflects the structure of wild-type LacY in the native membrane in the absence of sugar.

Fig. 1.

Single-Cys LacY mutants. Positions of Cys replacements are shown on the backbone of LacY (Protein Data Bank ID code 1PV7; www.pdb.org) viewed perpendicular to the membrane with the N-terminal helix bundle on the left and the C-terminal bundle on the right. Black spheres indicate single-Cys replacements on the cytoplasmic side of the sugar-binding site: 60, 67 (helix II), 279 (helix VIII), 329, and 331 (helix X); grey spheres indicate single-Cys replacements on the periplasmic side of the sugar-binding site: 31 (helix I), 42, 44, (helix II), 242, and 245 (helix VII). The light grey spheres at the apex of the inward-facing cavity represent TDG.

Fig. 2.

TMRM labeling of periplasmic N245C single-Cys LacY. (A and B) TMRM labeling in RSO membrane vesicles. (C and D), TMRM labeling in RSO membrane vesicles after addition of 2.0% DDM. (E and F), TMRM labeling of purified protein in DDM. Samples were incubated with 40 μM TMRM (A–D) or 4 μM TMRM (E and F), for the times indicated at 0 °C in the absence of sugar (−TDG; blue ◆), were preincubated with TDG (+TDG) for 10 min before addition of TMRM (green ▲), or TMRM and TDG were added simultaneously [+(TDG+TMRM); pink ■]. Samples then were subjected to minipurification with monovalent avidin, and the TMRM-labeled proteins were subjected to SDS/PAGE and assayed for fluorescence (lanes above dotted lines) and protein (lanes below dotted lines). The data then were treated semiquantitatively as described in Experimental Procedures by setting the 60-s point in the presence of TDG to 100%. Although not shown in A or B, when valinomycin (10 μM, final concentration) and nigericin (0.1 μM, final concentration) were added, no significant deviation from the pink curve (pink ■) shown in A was observed.

Fig. 3.

TMRM labeling of cytoplasmic S67C single-Cys LacY. (A) SDS/PAGE gels showing TMRM labeling in RSO membrane vesicles or with purified protein (fluorescence above dotted line; protein below). (B) Time course of TMRM labeling in RSO membrane vesicles. (C) Time course of TMRM labeling of purified mutant S67C in DDM. RSO vesicles with S67C LacY were labeled with 40 μM TMRM or purified S67C LacY in DDM with 4 μM TMRM for times indicated at 0 °C in the absence of TDG (−TDG), were preincubated with TDG for 10 min before addition of TMRM (+TDG), or TMRM and TDG were added simultaneously [+(TDG+TMRM)]. Purified proteins were subjected to SDS/PAGE; fluorescent and silver-stained protein bands were measured as described in Fig. 2 and Experimental Procedures. The data are plotted relative to the 20-s points in the absence of TDG. Blue ◆, no sugar added; green ▲, preincubated with TDG before addition of TMRM; pink ■, TDG and TMRM added simultaneously.

Fig. 4.

TMRM labeling of periplasmic single-Cys mutants in RSO membrane vesicles or as the purified proteins in DDM. Labeling of periplasmic single-Cys LacY mutants Q31C, K42C, D44C, Q242C, and N245C (Fig. 1) was performed with 40 μM TMRM (RSO membrane vesicles) or 4 μM TMRM (with purified proteins in DDM) for given times at 0 °C in the absence of TDG (−TDG; blue ◆) or after 10 min preincubation with TDG (+TDG; pink ■). Relative TMRM labeling rates were calculated as described in Experimental Procedures. The data are plotted relative to the 20-s points in the presence of TDG.

Fig. 5.

TMRM labeling of cytoplasmic single-Cys mutants in RSO membrane vesicles and as the purified proteins in DDM. Labeling of cytoplasmic single-Cys LacY mutants Q60C, S67C, A279C, L329C, and V331C (Fig. 1) was performed with 40 μM TMRM (RSO membrane vesicles) or 4 μM TMRM (with purified proteins in DDM) for given times at 0 °C in the absence of TDG (−TDG; blue ◆) or were preincubated for 10 min with TDG before addition of TMRM (+TDG; pink ■). Relative TMRM labeling rates were calculated as described in Experimental Procedures. The data are plotted relative to the 20-s points in the absence of TDG.