Crystal structure of lactose permease in complex with an affinity inactivator yields unique insight into sugar recognition

Abstract

Lactose permease of Escherichia coli (LacY) with a single-Cys residue in place of A122 (helix IV) transports galactopyranosides and is specifically inactivated by methanethiosulfonyl-galactopyranosides (MTS-gal), which behave as unique suicide substrates. In order to study the mechanism of inactivation more precisely, we solved the structure of single-Cys122 LacY in complex with covalently bound MTS-gal. This structure exhibits an inward-facing conformation similar to that observed previously with a slight narrowing of the cytoplasmic cavity. MTS-gal is bound covalently, forming a disulfide bond with C122 and positioned between R144 and W151. E269, a residue essential for binding, coordinates the C-4 hydroxyl of the galactopyranoside moiety. The location of the sugar is in accord with many biochemical studies.

Fig. 1.

MTS-gal chemistry (A and B) and transport (C). (A) Chemistry. Reaction with Cys residue. (B) Fluorescence detection of MTS-gal labeling. Top, UV fluorescence of LacY by SDS/PAGE; Bottom, the same gel stained with Coomassie Brilliant Blue to reveal protein loaded. (−) or (+) reflects the absence or presence, respectively, of MTS-gal during preincubation. Disappearance of fluorescence when single-Cys122 LacY was incubated with MTS-gal demonstrates complete labeling by the inactivator. (C) [³H]MTS-gal transport. RSO vesicles containing Cys-less LacY at 3 mg/mL were treated with 2 mM NEM to decrease background labeling. Transport was initiated by adding [³H]MTS-Gal in the presence of ascorbate and phenazine methosulfate to generate (interior negative). Reactions were terminated at a given time by rapid filtration. Data were corrected by subtracting a zero time control value of 1.3 nmol/mg protein.

Fig. 2.

(A) Structure of single-Cys122 LacY viewed from the membrane plane. The MTS-gal is shown as spheres colored by atom type with yellow carbons and bronze sulfurs. The N- and C-terminal bundles are colored blue and red, respectively. (B) Superposition of the C-terminal domains of C154G (PDB ID code 1PV7, gray) and single-Cys122 LacY showing slight displacement of the N-terminal bundle. As shown in this cytoplasmic view, the N-terminal domain of single-Cys122 LacY (blue) is displaced toward the C-terminal bundle (red) forming a narrower cavity. MTS-gal is represented as in A. Residues 190–220 and 400–417 have been removed for clarity. (C) Stereo view of the final 2mFo-DFc map (blue) of single-cys122 LacY structure contoured at 1σ. Helices are displayed as cartoon for ease of interpretation and the positions of atoms are shown as sticks. For validation purposes, we added on a naive mFo-DFc difference map (green) of the sugar-binding site contoured at 3σ. This later map has been calculated on a model where the MTS-gal was never introduced, showing an unbiased view of the sugar-binding site.

Fig. 3.

(A) MTS-gal binding site. Single-Cys122 LacY helices are colored as in Fig. 2. Side chains are displayed as sticks colored by atom type with yellow carbons and bronze sulfur atoms. The galactopyranosyl moiety of MTS-gal is equidistant between W151 and R144 stacking hydrophobically. The C-4 and C-6 OH groups of the galactopyranosyl ring are H-bonded (green dots) to E269. R144 is H-bonded to E126. (B) Space filling representation of A. (C) Superposition of sugar-binding sites displayed in stereo view. LacY in complex with TDG (PDB ID code 1PV7, gray) was superposed using Secondary Structure Matching on single-Cys122 LacY. The coordinations of 1PV7 to the substrate analogue TDG are depicted as gray dots.