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. 2007 Jul 31;104(31):12640-5.
doi: 10.1073/pnas.0700969104. Epub 2007 May 14.

Single-molecule FRET reveals sugar-induced conformational dynamics in LacY

Devdoot S Majumdar  1 , Irina Smirnova, Vladimir Kasho, Eyal Nir, Xiangxu Kong, Shimon Weiss, H Ronald Kaback
Affiliations

Single-molecule FRET reveals sugar-induced conformational dynamics in LacY

Devdoot S Majumdar et al. Proc Natl Acad Sci U S A. .

Abstract

The N- and C-terminal six-helix bundles of lactose permease (LacY) form a large internal cavity open on the cytoplasmic side and closed on the periplasmic side with a single sugar-binding site at the apex of the cavity near the middle of the molecule. During sugar/H(+) symport, an outward-facing cavity is thought to open with closing of the inward-facing cavity so that the sugar-binding site is alternately accessible to either face of the membrane. In this communication, single-molecule fluorescence (Förster) resonance energy transfer is used to test this model with wild-type LacY and a conformationally restricted mutant. Pairs of Cys residues at the ends of two helices on the cytoplasmic or periplasmic sides of wild-type LacY and the mutant were labeled with appropriate donor and acceptor fluorophores, single-molecule fluorescence resonance energy transfer was determined in the absence and presence of sugar, and distance changes were calculated. With wild-type LacY, binding of a galactopyranoside, but not a glucopyranoside, results in a decrease in distance on the cytoplasmic side and an increase in distance on the periplasmic side. In contrast, with the mutant, a more pronounced decrease in distance and in distance distribution is observed on the cytoplasmic side, but there is no change on the periplasmic side. The results are consistent with the alternating access model and indicate that the defect in the mutant is due to impaired ligand-induced flexibility on the periplasmic side.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structural modeling of LacY double-labeled with Alexa fluorophores at Cys pairs introduced at the cytoplasmic ends of helices III and XII (R73C/S401C) (Left) or the periplasmic ends of helices V and XI (I164C/R375C) (Right). Backbone of LacY rendered as ribbons rainbow colored from blue (helix 1) to red (helix 12) with hydrophilic cavity open to cytoplasmic side. Attached fluorophores colored in green (Alexa 488) or magenta (Alexa 647) and shown in space-filling representation. Estimated distances between Cα atoms of the Cys pairs and between centers of the fluorophores are 41 and 48 Å (cytoplasmic), and 32 and 61 Å (periplasmic), respectively.
Fig. 2.
Fig. 2.
Ligand-induced effects on the FRET distribution E* at the cytoplasmic side of LacY (R73C/S401C, helices III and XII). (A) Two-dimensional S–E* histograms corresponding to wild-type (A1–A3) and C154G mutant (A4–A6) LacY. Measurements for each construct were obtained in the absence of sugar (A1 and A4) and in the presence of 1 mM (saturating) NPG (A2 and A5) or 1 mM NPGlc (A3 and A6). (B and C) Comparison of sugar effects on normalized E* histograms from A for wild-type LacY (B) and C154G LacY (C). Gray bins, no sugar added; red line, 1 mM NPG; blue line, 1 mM NPGlc; broken lines, Gaussian fits to the data with the corrected E values given in Table 2.
Fig. 3.
Fig. 3.
Concentration dependence of ligand-induced effects on 〈E*〉 and ΔE* at the cytoplasmic side of LacY (R73C/S401C/C154G, helices III and XII). (A) E* distribution without added sugar (red) and with nine NPG concentrations increasing from 1 μM (orange) to 1,000 μM (purple). (B) Fractional change of the fitted Gaussian parameters of histograms from A plotted as a function of NPG concentration. Red, relative 〈E*〉 shift; black, relative ΔE* change. The maximum change in 〈E*〉 and ΔE* at saturating concentration of NPG (1 mM) corresponds to 1. Solid lines, hyperbolic fit to each data set; broken line, theoretical curve showing NPG binding to LacY with Kd of 38 μM as measured by stopped-flow (32).
Fig. 4.
Fig. 4.
Ligand-induced effects on the FRET distribution E* at the periplasmic side of LacY (I164C/S375C, helices V and XI). All details are as described in Fig. 2.
Fig. 5.
Fig. 5.
Cartoon model illustrating global conformational changes detected by sm-FRET upon sugar binding to wild-type LacY and C154G mutant. Without bound sugar (left) protein is in the protonated state with an inward-facing hydrophilic cavity. Without ligand, both molecules have multiple conformations on the cytoplasmic side (arrows). This conformation is the most energetically stable among multiple unliganded conformers. Binding of sugar induces a global conformational change in both wild-type LacY and C154G mutant resulting in closing of the inward-facing hydrophilic cavity. A cavity opens on the periplasmic in wild-type LacY allowing substrate to be released, and conformational heterogeneity increases after sugar binding. In contrast, the periplasmic cavity in C154G mutant does not form easily after sugar binding, corresponding to a reduced number of conformers in the ligand-bound state and restricted access for the sugar from periplasmic side.
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