Sugar binding induces an outward facing conformation of LacY

Abstract

According to x-ray structure, the lactose permease (LacY) is a monomer organized into N- and C-terminal six-helix bundles that form a deep internal cavity open on the cytoplasmic side with a single sugar-binding site at the apex. The periplasmic side of the molecule is closed. During sugar/H(+) symport, a cavity facing the periplasmic side is thought to open with closure of the inward-facing cytoplasmic cavity so that the sugar-binding site is alternately accessible to either face of the membrane. Double electron-electron resonance (DEER) is used here to measure interhelical distance changes induced by sugar binding to LacY. Nitroxide-labeled paired-Cys replacements were constructed at the ends of transmembrane helices on the cytoplasmic or periplasmic sides of wild-type LacY and in the conformationally restricted mutant Cys-154-->Gly. Distances were then determined in the presence of galactosidic or nongalactosidic sugars. Strikingly, specific binding causes conformational rearrangement on both sides of the molecule. On the cytoplasmic side, each of six nitroxide-labeled pairs exhibits decreased interspin distances ranging from 4 to 21 A. Conversely, on the periplasmic side, each of three spin-labeled pairs shows increased distances ranging from 4 to 14 A. Thus, the inward-facing cytoplasmic cavity closes, and a cleft opens on the tightly packed periplasmic side. In the Cys-154-->Gly mutant, sugar-induced closing is observed on the cytoplasmic face, but little or no change occurs on periplasmic side. The DEER measurements in conjunction with molecular modeling based on the x-ray structure provide strong support for the alternative access model and reveal a structure for the outward-facing conformer of LacY.

Fig. 1.

Disulfide-linked nitroxide chains are modeled on the LacY x-ray structure (PDB ID 1PV6) presented as rainbow colored ribbons from blue (helix I) to red (helix XII) with the hydrophilic cavity open to the cytoplasmic side. Nitroxides attached to the backbone of the protein are shown as balls and sticks. Interspin distances used for measurements are shown as the dashed lines. Cytoplasmic pairs R73C-S401C (helices III and XII), R73C-Q340C (helices III and X), S136C-Q340C (helices IV and X), S136C-S401C (helices IV and XII), N137C-Q340C (helices IV and X), and N137-S401C (helices IV and XII) are viewed from the side (A) and from cytoplasm (B). Periplasmic pairs V105C-T310C (helices IV and IX), I164C-T310C (helices V and IX), and I164C-S375C (helices V and XI) are viewed from side (A) and from periplasm (C). (D) Structure of the nitroxide side chain attached to the protein with indicated dihedral angles. Molscript (53) and Raster3D (54) were used in preparation of this illustration.

Fig. 2.

DEER characterization of sugar binding effects on interspin distances of nitroxide-labeled double-cysteine mutants located on the cytoplasmic side of LacY (helices III and XII or III and X). For each Cys pair listed on the left there are three panels: background corrected dipolar evolution data (echo amplitude recorded as a function of time) (A); dipolar spectra (Fourier transformation of the dipolar evolution data from A) (B); and distance distributions obtained by Tikhonov regularization (C). Data are shown for protein with no sugar bound (sucrose or NPGlc, green or blue, respectively) and with bound sugar (TDG or NPGal, pink or red lines, respectively). Broken lines in A and dotted lines in B are fits to the data by Tikhonov regularization. Plots in A and B are normalized by amplitude and shifted vertically for comparison. Plots in C show multi-Gaussian fits (black lines) demonstrating relative distributions of conformational populations (see also SI Table 2).

Fig. 3.

DEER characterization of sugar binding effects on interspin distances of nitroxide labeled double-cysteine mutants located on the cytoplasmic side of LacY (helices IV-X and IV-XII). Blue and red lines represent data obtained with NPGlc and NPGal, respectively. All other details are described in the legend for Fig. 2. Relative distributions of conformational populations calculated from multi-Gaussian fits are also presented in SI Table 3.

Fig. 4.

DEER characterization of sugar-binding effects on interspin distances of nitroxide-labeled double-cysteine mutants located on periplasmic side of LacY. Blue and red lines represent data obtained with NPGlc and NPGal, respectively. All other details are described in the legend for Fig. 2. Relative distributions of conformational populations calculated from multi-Gaussian fits are also presented in SI Table 4.

Fig. 5.

Molecular modeling of conformational changes in LacY upon sugar binding based on DEER distance measurements. Inward-facing conformation with no sugar bound (A), a transition state (B), and outward-facing conformation with bound sugar (C) correspond to three conformational populations of LacY observed in DEER measurements (see Figs. 2–4 and SI Tables 2–4). Transmembrane helices are rainbow colored from blue to red. Only two nitroxide pairs (cytoplasmic 73/401 and periplasmic 164/310) are shown as balls and sticks to demonstrate distance changes on cytoplasmic and periplasmic sides. The central loop between helices VI and VII is removed for clarity. Conformers with determined interspin distances are viewed normal to the membrane. (D–F) Space filling representations of conformers shown in A, B, and C, respectively, with helices II and VIII removed to demonstrate openings on cytoplasmic or periplasmic sides. The illustrations were prepared with Molscript (53), Raster3D (54), DINO (http://www.dino3d.org), and MSMS (55).