Structure of sugar-bound LacY

Abstract

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.

Keywords: X-ray structure; conformational change; induced fit; membrane protein; transport.

Fig. 1.

Side view of LacY G46W/G262W molecules A (Center) and B (Left) in an almost occluded, outward-facing conformation shown in green and gray ribbons, respectively. The two molecules in the asymmetric unit are shown adjacent to one another from the perspective of the membrane plane. The two molecules have a similar conformation with bound TDG, and the Trp replacements at positions 46 and 262 are shown. TDG and the Trp replacements are represented as spheres, with carbon atoms in magenta (for Trp) or orange (for TDG), oxygen atoms in red, nitrogen atoms in blue, and sulfur in yellow. Dashed lines depict the quasi twofold axes relating the N- and C-terminal helix bundles. (Right) The change in structure between LacY G46W/G262W (green; helices numbered) versus the apo WT structure (PDB ID code 2V8N, blue). The orientation matches chain A, and the alignment of the two structures is based on alignment of the N-terminal six-helix bundle of the apo structure onto the G46W/G262W.

Fig. 2.

Surface renditions of LacY G46W/G262W molecule A. (A) View from the periplasmic side showing TDG (green and red spheres) just visible within the molecule. (B) View from the cytoplasmic side, with the residues that form a zipper-like motif to seal that side portrayed as yellow sticks connected by a broken red line.

Fig. 3.

2mFo-DFc electron density in a B-factor–sharpened map contoured at 1σ (green mesh) at the sugar-binding site of LacY G46W/G262W, molecule A. The density is superimposed on the structure, which is shown as sticks, with carbon atoms in gold, oxygen atoms in red, and nitrogen atoms in blue. Broken lines represent putative hydrogen bonds.

Fig. 4.

The sugar-binding site in molecule B. (A) TDG is shown as green sticks, and side chains forming hydrogen bonds with TDG are in yellow. Broken lines represent likely hydrogen bonds. Ala122 and Cys148, which are close to TDG but do not make direct contact, are shown in light blue. Glu325 and Arg302 are transparent. For stereo views of the sugar-binding site in both chain A and chain B, see Fig. 6.

Fig. 5.

Crystal structure of single-Cys122 LacY with covalently bound MTS-Gal. (A) Helices are displayed as lines for ease of viewing, and side chains are shown as sticks. Side chains in yellow (Glu269 and Trp151) make direct contact with the galactopyranosyl ring of MTS-Gal covalently bound to a Cys at position 122. Side chains in gray are not sufficiently close to make direct contact with the galactopyranosyl ring. For a stereo view of the sugar-binding site, see Fig. 6. (B) Structure of single-Cys122 LacY with covalently bound MTS-Gal viewed from the side. Helices are depicted as rods, and MTS-Gal is shown as spheres colored by atom type with carbon ingreen. The water-filled central cavity open on the cytoplasmic side is colored light gray.

Fig. 6.

Stereo view of the sugar-binding site. TDG is shown as green stick model, and side chains forming hydrogen bonds with TDG are in yellow. Ala122 and Cys148, which are close to TDG but do not make direct contact, are shown in cyan. (Top) Molecule A with TDG. (Middle) Molecule B with TDG. (Bottom) LacY with covalently bound MTS-Gal (PDB ID code 2Y5Y).