Direct sugar binding to LacY measured by resonance energy transfer.

The lactose permease of Escherichia coli (LacY), a member of Major Facilitator Superfamily (1), is arguably the most intensively studied membrane transport protein (2, 3), and one of few with a known crystal structure (4). A proposed mechanism for lactose transport (3) includes ordered binding of an H+ and a galactopyranoside on one side of the membrane followed by a conformational change and ordered release of the sugar and the H+ on the opposite side. Although the X-ray structure of LacY reveals an inward-facing conformation, there is a body of data (3) supporting the concept that the transport cycle includes wide-spread conformational changes linked to the binding and release of sugar. Therefore, knowledge regarding individual steps in the transport mechanism is paramount to understanding the overall mechanism. Only a handful of side chains are essential for sugar recognition and binding (Fig. 1B). Arg144 (helix V) forms a bi-dentate H-bond with the O4 and O3 atoms of the galactopyranosyl ring (4), confirming the critical role of this residue in sugar binding and recognition (3). Glu126 (helix IV), another important residue for binding, is in close proximity to Arg144 and may interact with the O4, O5 or O6 atoms of the galactopyranosyl ring via water molecules. An aromatic residue at position 151 (helix V), preferably Trp, is irreplaceable for sugar binding (5), stacking hydrophobically with the galactopyranosyl ring (6, 7). Glu269 (helix VIII) in the C-terminal domain, which is also involved in H+ translocation, may interact with the O3 atom of the galactopyranosyl ring, forms a salt bridge with Arg144 and is in close proximity to Trp151 (4, 7-9). Figure 1 Location of Trp residues in the structure of C154G LacY with bound sugar (PDB ID: 1PV7). The structures is displayed with Pymol 0.98 (DeLano Scientific LLC, Inc.), and transmembrane helices are colored from blue (helix I) to red (helix XII). A. Overall ... Detection and quantification of sugar binding can be accomplished by flow dialysis or substrate protection against alkylation of Cys148 with maleimides (10-16). Cys148 is near the sugar-binding site in LacY, and sugar binding sterically blocks alkylation. Each of these techniques is a steady-state measurement, and involves use of radioactivity or fluorescence. Binding can also be detected and quantified by ligand-induced changes in the fluorescence of 2-(4’-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) with purified single-Cys V331C. In this case, the fluorophore is positioned far from sugar binding site and reflects a long-range conformational change resulting from sugar binding (11, 14, 15, 17). However, none of the methods have been used to measure kinetic parameters. In this paper we describe a novel method for determining and quantifying sugar binding by using the intrinsic Trp fluorescence of purified LacY. Formation of a donor-acceptor pair between Trp151 and α-NPG or 6’-(N-dansyl)aminohexyl-1-thio-β-d-galactopyranoside (Dns6Gal) in the sugar-binding site results in Forster resonance energy transfer (FRET), which can be measured in the steady-state or by stopped-flow. In addition, the time course of a ligand-induced conformational change is measured with MIANS-labeled V331C LacY. The results are consistent with a mechanism in which a relatively rapid sugar-binding step is followed by a slower conformational change.