Clogging the periplasmic pathway in LacY.

The lactose permease of Escherichia coli (LacY), a member of the Major Facilitator Superfamily of membrane transport proteins, couples the stoichiometric translocation of a galactoside and an H+ (reviewed in 1, 2). In this manner, LacY is able to utilize the free energy stored in an electrochemical H+ gradient (ΔμH +; interior negative and/or alkaline) to drive accumulation of galactosidic sugars against a concentration gradient. Conversely, in the absence of ΔμH +, LacY utilizes the free energy released from downhill translocation of galactosides to drive uphill translocation of H+ with generation of ΔμH +, the polarity of which depends on the direction of the sugar gradient. LacY has been solubilized, purified and reconstituted into proteoliopsomes in a fully functional state (3). X-ray crystal structures of a conformationally restricted mutant (4–9) have been solved in an inward-facing conformation (10, 11), and an X-ray crystal structure of the wild type has the same global fold (12, 13). Each structure exhibits twelve transmembrane helices organized into two pseudo-symmetrical six α-helical bundles surrounding a large interior hydrophilic cavity open to the cytoplasm, which represents an inward-facing conformation. The sugar-binding site and the residues involved in H+ translocation are at the approximate middle of the molecule and distributed such that the side chains important for sugar recognition are in the N-terminal helix bundle, while most of the side chains important for H+ translocation are in the C-terminal bundle. Strikingly, the periplasmic side of LacY is tightly packed, and the sugar-binding site is inaccessible from the periplasm. Wild-type LacY is dynamic, ligand binding is entropic (7) and induces widespread conformational changes (1, 2, 7, 14, 15). Site-directed alkylation (SDA) (reviewed in 16, 17, 18), single molecule fluorescence resonance energy transfer (smFRET) (8), double electron-electron resonance (DEER) (9) and cross-linking studies (19) provide strong evidence that sugar binding increases the open probability of a relatively wide hydrophilic cleft on the periplasmic side of LacY. Moreover, this cleft must close, as well as open, for translocation of sugar across the membrane to occur (19). Remarkably, despite multiple independent lines of evidence for a hydrophilic pathway that opens upon sugar binding in wild-type LacY, as well as findings demonstrating that the periplasmic pathway in the C154G mutant is paralyzed in the open conformation (8, 9, 18), all X-ray structures of LacY exhibit the same inward-facing conformation. Therefore, it is likely that the crystallization process selects a single conformer of LacY that is in the lowest free energy state. In this communication, single-Cys replacements in helix VII, one face of which lines the periplasmic pathway (Fig. 1), were labeled with a given thiol reagent (Fig. 2) in the presence of β-D-galactopyranosyl 1-thio-β-D-galactopyranoside (TDG) in order to ensure high accessibility/reactivity of the Cys replacement (16–18). Transport activity or sugar binding to the modified proteins, which were reacted to completion, was then studied in right-side-out (RSO) membrane vesicle preparations that are quantitatively RSO (20–26) and in which each vesicle is active with respect to transport (27). Inhibition of transport activity correlates approximately with the size of the thiol reagent, but shape and/or charge are also probably important. In any case, sugar binding is unaffected despite complete inactivation of transport with the largest thiol reagent. The findings are consistent with the interpretation that when the periplasmic pathway is prevented from closing, the sugar-binding site remains accessible from periplasmic side, but the transport cycle is arrested. Figure 1 Q241, Q242 and N245 line the potential periplasmic pathway of LacY. A. LacY is viewed perpendicular to the membrane with the N-terminal helix bundle on the left and the C-terminal bundle on the right. B. LacY is viewed from periplasmic side of the membrane ... Figure 2 Thiol reagents