Trp replacements for tightly interacting Gly–Gly pairs in LacY stabilize an outward-facing conformation

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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...

mentioning

confidence: 61%

Real-time conformational changes in LacY

Смирнова¹,

Kasho²,

Kaback

2014

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“…The G46W/G262W mutant of LacY described here behaves as if it is arrested in an outward-open conformation with diffusion-limited access to the binding site based on biochemical and spectroscopic measurements (44). The X-ray crystal structure has bound substrate and as a result is almost occluded and still is partially open to the periplasmic side.…”

Section: Discussionmentioning

confidence: 90%

“…Moreover, site-directed alkylation and stopped-flow binding kinetics indicate that the G46W/ G262W mutant is open on the periplasmic side (open outward). In addition, the detergent-solubilized mutant exhibits much greater thermal stability than WT LacY (44).…”

Section: Significancementioning

confidence: 99%

See 1 more Smart Citation

Structure of sugar-bound LacY

Johri

Kasho

Смирнова

et al. 2014

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118

167

Here we describe the X-ray crystal structure of a double-Trp mutant (Gly46→Trp/Gly262→Trp) of the lactose permease of Escherichia coli (LacY) with a bound, high-affinity lactose analog. Although thought to be arrested in an open-outward conformation, the structure is almost occluded and is partially open to the periplasmic side; the cytoplasmic side is tightly sealed. Surprisingly, the opening on the periplasmic side is sufficiently narrow that sugar cannot get in or out of the binding site. Clearly defined density for a bound sugar is observed at the apex of the almost occluded cavity in the middle of the protein, and the side chains shown to ligate the galactopyranoside strongly confirm more than two decades of biochemical and spectroscopic findings. Comparison of the current structure with a previous structure of LacY with a covalently bound inactivator suggests that the galactopyranoside must be fully ligated to induce an occluded conformation. We conclude that protonated LacY binds D-galactopyranosides specifically, inducing an occluded state that can open to either side of the membrane.T he lactose permease of Escherichia coli (LacY), a paradigm for the major facilitator superfamily (MFS), binds and catalyzes transport of D-galactose and D-galactopyranosides specifically with an H + (1, 2). In contrast, LacY does not recognize D-glucose or D-glucopyranosides, which differ only in the orientation of the C4-OH of the pyranosyl ring. By using the free energy released from the energetically downhill movement of H + in response to the electrochemical H + gradient (Δμ H +), LacY catalyzes the uphill (active) transport of galactosides against a concentration gradient. Because coupling between sugar and H + translocation is obligatory, in the absence of Δμ H +, LacY also can transduce the energy released from the downhill transport of sugar to drive uphill H + transport with the generation of Δμ H +, the polarity of which depends upon the direction of the sugar gradient.It also has been shown that LacY binds sugar with a pK a of ∼10.5 and that sugar binding does not induce a change in ambient pH; both findings indicate that the protein is protonated over the physiological range of pH (3-5). These observations and many others (1, 2) provide evidence for an ordered kinetic mechanism in which protonation precedes galactoside binding on one side of the membrane and follows sugar dissociation on the other side. Recent considerations (6) suggest that a similar ordered mechanism may be common to other members of the MFS.Because equilibrium exchange and counterflow are unaffected by imposition of Δμ H +, it is apparent that the alternating accessibility of sugar-and H + -binding sites to either side of the membrane is the result of sugar binding and dissociation and not of Δμ H + (reviewed in refs. 1 and 2). Moreover, downhill lactose/ H + symport from a high to a low lactose concentration exhibits a primary deuterium isotope effect that is not observed for Δμ H +-driven lactose/H + symport, equilibrium exchange, or count...

“…When Gly46 (helix II) and Gly262 (helix VIII) are replaced with bulky Trp residues (Fig. 2), transport activity is abrogated with little or no effect on galactoside affinity, but markedly increased accessibility of galactoside to the binding site is observed, indicating that the G46W/G262W mutant is open on the periplasmic side (11). Moreover, sitedirected alkylation and stopped-flow binding kinetics indicate that the G46W/G262W mutant is physically open on the periplasmic side (an outward-open conformation).…”

Section: Structural Evidence For An Occluded Intermediatementioning

confidence: 99%

A chemiosmotic mechanism of symport

Kaback

2015

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121

Lactose permease (LacY), a paradigm for the largest family of membrane transport proteins, catalyzes the coupled translocation of a galactoside and an H + across the Escherichia coli membrane (galactoside/H + symport). Initial X-ray structures reveal N-and C-terminal domains, each with six largely irregular transmembrane helices surrounding an aqueous cavity open to the cytoplasm. Recently, a structure with a narrow periplasmic opening and an occluded galactoside was obtained, confirming many observations and indicating that sugar binding involves induced fit. LacY catalyzes symport by an alternating access mechanism. Experimental findings garnered over 45 y indicate the following: (i) The limiting step for lactose/H + symport in the absence of the H + electrochemical gradient (Δμ̃H+) is deprotonation, whereas in the presence of Δμ̃H+, the limiting step is opening of apo LacY on the other side of the membrane; (ii) LacY must be protonated to bind galactoside (the pK for binding is ∼10.5); (iii) galactoside binding and dissociation, not Δμ̃H+, are the driving forces for alternating access; (iv) galactoside binding involves induced fit, causing transition to an occluded intermediate that undergoes alternating access; (v) galactoside dissociates, releasing the energy of binding; and (vi) Arg302 comes into proximity with protonated Glu325, causing deprotonation. Accumulation of galactoside against a concentration gradient does not involve a change in K d for sugar on either side of the membrane, but the pK a (the affinity for H + ) decreases markedly. Thus, transport is driven chemiosmotically but, contrary to expectation, ΔμH+ acts kinetically to control the rate of the process.The lactose permease of Escherichia coli (LacY) specifically binds and catalyzes symport of D-Gal and D-galactopyranosides with an H + (galactoside/H + symport), but it does not recognize the analogous glucopyranosides, which differ only in the orientation of the C4-OH of the pyranosyl ring (reviewed in refs. 1, 2). Typical of many major facilitator superfamily (MFS) members, LacY couples the free energy released from downhill translocation of H + in response to an H + electrochemical gradient (ΔμH+) to drive accumulation of galactopyranosides against a concentration gradient. Because coupling between sugar and H + translocation is obligatory, in the absence of Δμ̃H+, LacY can also transduce the energy released from the downhill transport of sugar to drive uphill H + transport with the generation of Δμ̃H+, the polarity of which depends upon the direction of the sugar gradient. However, the mechanism by which this so-called "chemiosmotic" process occurs remains obscure. This contribution aims at clarifying the specific steps underpinning the mechanism of galactoside/ H + symport.