It takes two to tango: The dance of the permease

Abstract

The lactose permease (LacY) of Escherichia coli is the prototype of the major facilitator superfamily, one of the largest families of membrane transport proteins. Structurally, two pseudo-symmetrical six-helix bundles surround a large internal aqueous cavity. Single binding sites for galactoside and H⁺ are positioned at the approximate center of LacY halfway through the membrane at the apex of the internal cavity. These features enable LacY to function by an alternating-access mechanism that can catalyze galactoside/H⁺ symport in either direction across the cytoplasmic membrane. The H⁺-binding site is fully protonated under physiological conditions, and subsequent sugar binding causes transition of the ternary complex to an occluded intermediate that can open to either side of the membrane. We review the structural and functional evidence that has provided new insight into the mechanism by which LacY achieves active transport against a concentration gradient.

Figure 1.

X-ray crystal structure of WT LacY (PDB accession no. 2V8N). LacY molecule in an inward-facing conformation. In the ribbon representations of LacY (a and b), the N- and C-terminal six transmembrane helices are colored in blue and green, and the middle cytoplasmic loop is colored yellow. (a) Viewed perpendicular to the membrane with helices V and VIII in front. The sidedness of the membrane is labeled. (b) Cytoplasmic view normal to the membrane. Helices are labeled in roman numerals. (c–f) Calculated electrostatic surface potential of LacY molecule. The scale indicates color-coded values of the electrostatic potentials (kT/e). (c and d) Viewed perpendicular to the membrane. (e and f) Viewed parallel to the membrane from cytoplasmic and periplasmic sides.

Figure 2.

Mutant G46W/G262W and galactoside binding. Crystal structures of the LacY G46W/G262W mutant in a partially outward-facing occluded conformation with bound α-NPG (PDB accession no. 4ZYR) or TDG (PDB accession no. 4OAA). Both lactose analogues are shown in black. (a) Slab view of a surface representation of the LacY G46W/G262W molecule revealed by crystallography (PDB accession no. 4ZYR). An occluded α-NPG molecule is shown as sticks. (b) Binding interactions of α-NPG with LacY. (c) Binding interactions of TDG with LacY. The helices of LacY are colored in rainbow and labeled with roman numerals. Side chains that directly contact the sugar are shown as sticks. Dotted lines indicate interactions between the sugar and the protein, as well as between residues. The number in red indicates the hydroxyl group position in galactopyranosyl ring.

Figure 3.

Transport reactions catalyzed by LacY. (a) Active transport. The electrochemical H⁺ gradient ($\Delta {\tilde{\mu }}_{{\mathrm{H}}^{+}}$) across the cytoplasmic membrane of E. coli is generated by efflux of H⁺ via the respiratory chain or through the hydrolytic activity of F₁F_o ATPase. Free energy released from the energetically favored downhill movement of H⁺ catalyzed by LacY (yellow) is converted to the uphill accumulation of lactose as indicated by the direction of the arrows and font size. (b) Influx. (c) Efflux. Energetically downhill lactose transport generates $\Delta {\tilde{\mu }}_{{\mathrm{H}}^{+}},$ the polarity of which depends upon the direction of lactose concentration gradient (influx generates a $\Delta {\tilde{\mu }}_{{\mathrm{H}}^{+}}$ that is interior positive and acid; efflux generates a $\Delta {\tilde{\mu }}_{{\mathrm{H}}^{+}}$ that is interior negative and alkaline). (d) Equilibrium exchange. At equal intra- and extracellular lactose concentrations, lactose exchange across the membrane is catalyzed by protonated LacY. (e) Counterflow. At a high intracellular and low extracellular lactose concentrations, transient influx of radiolabeled external lactose or “counterflow” across the membrane is observed. The reaction is catalyzed by protonated LacY. Lac, lactose; SH, substrate in a reduced form.

Figure 4.

pK_a of Glu325 in LacY. The pH dependence of Δ-IR intensity change at 1,742 cm⁻¹ was measured with the LacY G46W/G262W mutant in the absence or presence of α-NPG (filled red circles) or E325A LacY (open red circles). The K_d values for α-NPG binding to WT LacY (filled green circles), E325A LacY (open green circles), or LacY G46W/G262W mutant (filled cyan circles) were calculated as the ratio of rate constants (k_off/k_on) measured by stopped-flow fluorescence.

Figure 5.

Position of Glu325 in WT LacY (PDB accession no. 2V8N). LacY is presented as rainbow-colored backbone (from blue to red for helices I to XII) with a hydrophilic cavity open to the cytoplasmic side. The side chain of Glu325 in helix X is shown as spheres. The area around Glu325 is enlarged with the hydrophobic environment displayed as a space-filled cartoon (cyan).

Figure 6.

Environment of Glu325. Crystal structure of the LacY G46W/G262W mutant in an outward-facing partially occluded conformation (PDB accession no. 4OAA). Helices of LacY are colored in rainbow and labeled with roman numerals. Dotted lines indicate polar/charged side chains/OH groups that are in close proximity and may form salt-bridge/H-bond interactions. TDG is shown in black, and C₃ OH is indicated. Glu325 is in helix X.

Figure 7.

The H⁺-lactose symport mechanism. There are eight kinetic steps as indicated. H⁺ and galactoside bind in the middle of the molecule, and LacY moves around the binding sites to release the sugar and the H⁺ by an ordered mechanism on either side of the membrane as indicated by the arrows. The alternating-access process involves the shaded steps only (2–5). S, substrate; H⁺, proton.