Structural determination of wild-type lactose permease

Abstract

Here we describe an x-ray structure of wild-type lactose permease (LacY) from Escherichia coli determined by manipulating phospholipid content during crystallization. The structure exhibits the same global fold as the previous x-ray structures of a mutant that binds sugar but cannot catalyze translocation across the membrane. LacY is organized into two six-helix bundles with twofold pseudosymmetry separated by a large interior hydrophilic cavity open only to the cytoplasmic side and containing the side chains important for sugar and H(+) binding. To initiate transport, binding of sugar and/or an H(+) electrochemical gradient increases the probability of opening on the periplasmic side. Because the inward-facing conformation represents the lowest free-energy state, the rate-limiting step for transport may be the conformational change leading to the outward-facing conformation.

Fig. 1.

Cartoon representation of the overall structure of wild-type LacY. (A) The residues involved in substrate binding [Glu-126 (helix IV), Arg-144, Cys-148, and Trp-151 (helix V)] are shown as pink sticks, and those involved in H⁺ translocation [Tyr-236 (helix VII), Arg-302 (helix IX), His-322, and Glu-325 (helix X)] are shown in yellow. The dual-function residue Glu-269 (helix VIII) is shown in green. Cys-154 is shown as van der Waals spheres (yellow). Pro-28, Pro-31, Ile-32, and His-35 on helix I (yellow surface) and Gln-241, Gln-242, Ala-244, Asn-245, and Thr-248 on helix VII (cyan surface) form a periplasmic gateway. The black color represents the back side of the surfaces. (B) View from cytoplasmic side with Ile-32 (helix I) close to Asn-245 (helix VII) shown as sticks with transparent van der Waals spheres (green). (C) View from periplasmic side with Ile-32 close to Asn-245.

Fig. 2.

Substrate-binding site. (A) 2F_o − F_c electron density (blue) contoured at 1 σ around residues (gray) located in the sugar-binding site. (B) Superposition of sugar-binding residues from wild-type (gray) and C154G LacY (pink; 2cfq). Salt bridges (Arg-144/Glu-126) are shown as black dotted lines, and an H bond (Glu-269–Trp-151) in C154G is shown as a pink dotted line.

Fig. 3.

Cys-154. (A) F_o − F_c electron density map (green) contoured at 4 σ around residue Cys-154. The map was calculated during refinement before residue 154 was changed to Cys from Gly. The map pinpoints the position of the Cys-154 sulfur. (B) The side chains of Cys-154 (yellow, helix V), Phe-21/Gly-24 (gray, helix I), and backbone carbonyl oxygen (red), as well as nitrogen (blue), are shown as sticks surrounded by van der Waals dots. The H bonds are shown as broken dots.