Real-time conformational changes in LacY

Abstract

Galactoside/H + symport across the cytoplasmic membrane of Escherichia coli is catalyzed by lactose permease (LacY), which uses an alternating access mechanism with opening and closing of deep cavities on the periplasmic and cytoplasmic sides. In this study, conformational changes in LacY initiated by galactoside binding were monitored in real time by Trp quenching/unquenching of bimane, a small fluorophore covalently attached to the protein.Rates of change in bimane fluorescence on either side of LacY were measured by stopped flow with LacY in detergent or in proteoliposomes and were compared with rates of galactoside binding. With LacY in proteoliposomes, the periplasmic cavity is tightly sealed and the substrate-binding rate is limited by the rate of opening of this cavity. Rates of opening, measured as unquenching of bimane fluorescence, are 20-30 s −1 , independent of sugar concentration and essentially the same in detergent or in proteoliposomes. On the cytoplasmic side of LacY in proteoliposomes, slow bimane quenching (i.e., closing of the cavity) is observed at a rate that is also independent of sugar concentration and similar to the rate of sugar binding from the periplasmic side. Therefore, opening of the periplasmic cavity not only limits access of sugar to the binding site of LacY but also controls the rate of closing of the cytoplasmic cavity. LacY is organized into two pseudosymmetrical bundles, each containing six transmembrane helices, most of which are irregular, with the N and C termini on the cytoplasmic side of the membrane. WT LacY and a conformationally restricted mutant exhibit an inward-facing conformation with a tightly sealed periplasmic side and a water-filled cavity open to the cytoplasm (6-9), which is the conformation present in the membrane in the absence of sugar (10). Another conformation has been observed recently with a double-Trp mutant (11) that exhibits an occluded galactoside molecule with a narrow opening on the periplasmic side and a tightly sealed cytoplasmic aspect (12) (Fig. 1A).LacY is highly dynamic and exhibits multiple conformations at any given time, and galactoside binding triggers a shift between conformers (1). Thus, measurement of interspin distances with nitroxide-labeled Cys pairs in LacY reveals that sugar binding induces a decrease in distances on the cytoplasmic side and a corresponding increase in distances on the periplasmic side (13,14). Site-directed alkylation of single Cys LacY mutants in either right-side-out membrane vesicles (15, 16) or dodecyl-β-D-maltopyranoside (DDM) micelles (10), as well as single-molecule fluorescence (17) and thiol cross-linking (18), also indicates that sugar binding increases the probability of opening on the periplasmic side and closing on the cytoplasmic side.Recently, conformational changes in LacY have been studied dynamically by site-directed Trp fluorescence quenching by a protonated His or Lys residue (19). Sugar binding leads to unquenching of Trp fluorescence in periplasmic LacY mutants N245W (helix VII) or F378...

Outward-facing conformers of LacY stabilized by nanobodies

“…Sugar-binding rates with WT LacY in PLs measured by Trp151→4-nitrophenyl-α-D-galactopyranoside (NPG) FRET are independent of sugar concentration, whereas the mutant with an open periplasmic cavity is characterized by a linear concentration dependence of sugar binding rates with k on of ∼10 μM −1 ·s −1 (18,20), which approximates diffusion controlled access to the binding site (21). Therefore, with WT LacY embedded in PLs, the periplasmic side is sealed, and substrate binding is limited by opening of the periplasmic cavity at a rate of 20-30 s −1 (19). This rate is very similar to the turnover number of WT LacY in right-side-out (RSO) membrane vesicles or reconstituted PLs (22) and is consistent with the notion that opening of the periplasmic cavity may be a limiting step in the overall transport mechanism.…”

“…Trp-induced bimane unquenching allows direct demonstration of opening of periplasmic cavity in LacY (19). Thus, bimane-labeled mutant F29W/G262C exhibits unquenching of bimane fluorescence after addition of sugar, indicating opening of the periplasmic cavity and even greater unquenching is observed after addition of Nb 9036 (Fig.…”

“…Numerous studies confirm that this conformation prevails in the absence of sugar (12-16). Recently, however, the X-ray structure of double-Trp mutant G46W/G262W with bound sugar reveals a conformation with a narrowly open periplasmic pathway and a tightly sealed cytoplasmic side (PDB ID code 4OAA) (17), thereby providing structural evidence that an intermediate occluded conformation occurs between the outward-and inwardfacing conformations in the transport cycle.Rates of opening/closing of periplasmic and cytoplasmic cavities have been determined in real time from changes in fluorescence of Trp or attached fluorophores with LacY either in detergent micelles or in reconstituted proteoliposomes (PLs) (15,18,19). Sugar-binding rates with WT LacY in PLs measured by Trp151→4-nitrophenyl-α-D-galactopyranoside (NPG) FRET are independent of sugar concentration, whereas the mutant with an open periplasmic cavity is characterized by a linear concentration dependence of sugar binding rates with k on of ∼10 μM −1 ·s −1 (18,20), which approximates diffusion controlled access to the binding site (21).…”

“…Rates of opening/closing of periplasmic and cytoplasmic cavities have been determined in real time from changes in fluorescence of Trp or attached fluorophores with LacY either in detergent micelles or in reconstituted proteoliposomes (PLs) (15,18,19). Sugar-binding rates with WT LacY in PLs measured by Trp151→4-nitrophenyl-α-D-galactopyranoside (NPG) FRET are independent of sugar concentration, whereas the mutant with an open periplasmic cavity is characterized by a linear concentration dependence of sugar binding rates with k on of ∼10 μM −1 ·s −1 (18,20), which approximates diffusion controlled access to the binding site (21).…”

“…4A). Therefore, interaction of the Nbs with LacY was studied by site-directed Trp-induced fluorescence quenching of bimane-or ATTO655-labeled LacY (19,37,38). WT LacY with a Cys replacement on the periplasmic side (I32C) labeled with bimane or ATTO655 exhibits a decrease in the fluorescence emission of either fluorophore upon addition of Nbs (Fig.…”