Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacY

Abstract

Cation-coupled active transport is an essential cellular process found ubiquitously in all living organisms. Here, we present two novel ligand-free X-ray structures of the lactose permease (LacY) of Escherichia coli determined at acidic and neutral pH, and propose a model for the mechanism of coupling between lactose and H+ translocation. No sugar-binding site is observed in the absence of ligand, and deprotonation of the key residue Glu269 is associated with ligand binding. Thus, substrate induces formation of the sugar-binding site, as well as the initial step in H+ transduction.

Figure 1

Overall structure of C154G LacY in the absence of ligand. LacY consists of 12 transmembrane helices organized in two pseudo-symmetrical α-helical bundles. The N- and C-terminal 6-helix domains form an internal cavity open to the cytoplasmic side. In the ligand-free structure at pH 6.5, the residues involved in substrate binding (Glu¹²⁶ (helix IV), Arg¹⁴⁴ and Trp¹⁵¹ (helix V)) are shown in yellow, and those involved in H⁺ translocation [Tyr²³⁶ (helix VII), Arg³⁰² (helix IX), His³²² and Glu³²⁵ (helix X)] are shown in pink. The dual-function residue Glu²⁶⁹ (helix VIII) is shown in cyan. (A) Side view; (B) cytoplasmic view.

Figure 2

Re-arrangement of the active site in the absence and presence of sugar. (A) Cytoplasmic stereo view of the sugar-binding site from superimposed crystal structures. The TDG-bound structure (1pv7) is shown in blue (3); ligand-free structures are shown in green (pH 5.6) and in yellow (pH 6.5). (B) Residues involved in sugar binding are shown with a 2F_o−F_c sigmaA weighted electron density map from the structure obtained at pH 6.5. (C) Stereo view of Glu²⁶⁹ in a relatively hydrophobic environment surrounded by residues Trp¹⁵¹, Cys¹⁴⁸, Asn²⁷², Ala²⁷³ and Met³²³.

Figure 3

Stereo view of residues involved in H⁺ translocation network. (A) Residues involved in H⁺ translocation in the TDG-bound structure (blue; 1pv7) and ligand-free structure (green) at pH 5.6. (B) Side-chains involved in H⁺ translocation at pH 5.6 in the absence of ligand shown with a 2F_o−F_c sigmaA weighted electron density map.

Figure 4

Proposed model for the initial step in coupling between sugar and H⁺ translocation. H-bonds are represented by broken lines; charge pairs by solid lines. The proton is represented by a white sphere and the water by a turquoise sphere. (A) Glu²⁶⁹ is protonated and close to Trp¹⁵¹ to form a platform in the absence of substrate. Arg¹⁴⁴ forms a salt bridge with Glu²⁶⁹. (B) The Glu²⁶⁹/Trp¹⁵¹ platform orients the galactopyranosyl ring to allow specific interactions with Arg¹⁴⁴, Glu¹²⁶ and Glu²⁶⁹. (C) Arg¹⁴⁴ moves towards the galactopyranoside ring to form a bi-dentate H-bond with the O₄ and O₃ positions. Glu²⁶⁹ moves into a hydrophilic environment, deprotonates and interacts with the O₃ position of the sugar, as well as a salt bridge with Arg¹⁴⁴. (D) The H⁺ is transferred to His³²² via water.

Figure 5

An updated influx mechanism for lactose/H⁺ symport. See main text for details. The proton (H⁺) and the substrate (S) are shown with red and green, respectively. Charge pairs are indicated by solid lines.