Lessons from lactose permease

Abstract

An X-ray structure of the lactose permease of Escherichia coli (LacY) in an inward-facing conformation has been solved. LacY contains N- and C-terminal domains, each with six transmembrane helices, positioned pseudosymmetrically. Ligand is bound at the apex of a hydrophilic cavity in the approximate middle of the molecule. Residues involved in substrate binding and H+ translocation are aligned parallel to the membrane at the same level and may be exposed to a water-filled cavity in both the inward- and outward-facing conformations, thereby allowing both sugar and H+ release directly into either cavity. These structural features may explain why LacY catalyzes galactoside/H+ symport in both directions utilizing the same residues. A working model for the mechanism is presented that involves alternating access of both the sugar- and H+-binding sites to either side of the membrane.

Figure 1

Lactose/H⁺ symport. In the absence of substrate, LacY does not translocate H⁺ in the presence of Δμ̄_H+. (a) Free energy released from the downhill movement of H⁺ is coupled to the uphill accumulation of lactose. (b, c) Substrate gradients generate electrochemical H⁺ gradients, the polarity of which depends upon the direction of the substrate concentration gradient.

Figure 2

Secondary structure model of LacY derived from the X-ray structure. The helices (rectangles) traverse the membrane in a zigzag fashion connected by hydrophilic domains external to the membrane. The helices are drawn to scale according to the X-ray structure, and dark green areas represent the portions of the helices that are within the membrane. The loops depict connectivity only. Residues at the kinks in the transmembrane helical domains are shown as light blue rectangles; residues in black rectangles are involved in substrate binding; residues in red are involved in H⁺ translocation. Glu-269 (black rectangle bordered in red) is involved in both substrate binding and H⁺ translocation. Salt-bridged residues are shown as orange rectangles. The large hydrophilic cavity is designated by an inverted yellow triangle, and sugar is depicted by two green circles, with N and C representing those moieties that interact with the N- and C-terminal halves of LacY, respectively.

Figure 3

The figure is based on the C154G mutant structure with bound TDG. (a) Ribbon representation of LacY viewed parallel to the membrane. The 12 transmembrane helices from the N and C termini are colored from dark blue (N-terminal bundles) to red (C-terminal bundles), and TDG is represented by black spheres. (b) Ribbon representation of LacY viewed along the membrane normal from the cytoplasmic side. For clarity, the loop regions have been omitted. The color scheme is the same as in panel (a), and the 12 transmembrane helices are labeled with Roman numerals. Reproduced, with permission, from Reference .

Figure 4

Substrate-binding site of LacY. Possible H-bonds and salt bridges are represented by broken blue lines. (a) Residues involved in TDG binding viewed along the membrane normal from the cytoplasmic side. TDG is depicted as a stick model. (b) Close up of the N-terminal domain of the TDG-binding site. Reproduced, with permission, from Reference .

Figure 5

Kinetic scheme for galactoside/H⁺ symport, exchange, and counterflow. Y represents LacY, and S is substrate (lactose). Steps involved in exchange and counterflow (steps 2,3, and 4) are in orange. During downhill symport in the absence of Δμ̄_H+, the rate-limiting step is deprotonation (step 1 or 5); in the presence of Δμ̄_H+, dissociation of sugar is rate limiting (step 2 or 4).

Figure 6

Residues involved in H⁺ translocation and coupling. H-bonds are represented by black broken lines. (a) View parallel to the membrane. (b) View along the membrane normal from the cytoplasmic side. Reproduced, with permission, from Reference .

Figure 7

Configuration of residues involved in sugar binding (green) and H⁺ translocation (orange). (a) Viewed parallel to the membrane. (b) Viewed along the membrane normal from the cytoplasmic side.

Figure 8

A postulated mechanism for lactose/H⁺ symport. Key residues are labeled and charge pairs are represented as solid black lines; H-bonds are depicted as broken black lines. The H⁺ and the substrate are shown in red and green, respectively.

Figure 9

Possible structural changes between inward- and outward-facing conformations. Transmembrane helices in the N- and C-terminal halves are shown as blue and red cyclinders, respectively. (a) Inward-facing conformation (i.e., the crystal structure) viewed parallel to the membrane. Cys-replacement mutants of residues colored in yellow exhibit increased reactivity with NEM upon substrate binding. (b) Suggested model for outward-facing conformation based on chemical modification and thiol cross-linking. The model was obtained by applying a relatively rigid body rotation of ∼60° (around the axis passing near TDG parallel to the membrane) to the N- and C-terminal domains. Reproduced, with permission, from Reference .

Figure 10

Effect of Δμ̄_H+ of opposite polarities on substrate translocation in RSO or ISO vesicles. (a) Δμ̄_H+ with RSO vesicles (ΔΨ, interior negative); substrate is accumulated. (b) Δμ̄_H+ in ISO vesicles (interior positive and acid); substrate effluxes from the vesicles. Reproduced, with permission, from Reference .