Monitoring Conformational Rearrangements in the Substrate-binding Site of a Membrane Transport Protein by Mass Spectrometry

Abstract

Combined biochemical, biophysical, and crystallographic studies on the lactose permease of Escherichia coli suggest that Arg-144 (helix V) forms a salt bridge with Glu-126 (helix IV), which is broken during substrate binding, thereby permitting the guanidino group to form a bidentate H-bond with the C-4 and C-3 O atoms of the galactopyranosyl moiety and an H-bond with Glu-269 (helix VIII). To examine the relative interaction of Arg-144 with these two potential salt bridge partners (Glu-126 and Glu-269) in the absence of substrate, the covalent modification of the guanidino group was monitored with the Arg-specific reagent butane-2,3-dione using electrospray ionization mass spectrometry. In a functional background, the reactivity of Arg-144 with butane-2,3-dione is low (ϳ25%) and is reduced by a factor of approximately 2 by preincubation with ligand. Interestingly, although replacement of Glu-126 with Ala results in a 3-fold increase in the reactivity of Arg-144, replacement of Glu-269 with Ala elicits virtually no effect. Taken together, these results suggest that in the absence of substrate the interaction between Arg-144 and Glu-126 is much stronger than the interaction with Glu-269, supporting the contention that sugar recognition leads to rearrangement of charge-paired residues essential for sugar binding. The major facilitator superfamily (MFS)1 is one of the largest families of membrane transport proteins found in archaeal, bacterial, and eukaryotic cell membranes (1) and contains over 1000 members, some of which are clinically relevant (e.g. the glucose transporters). As revealed by phylogenetic analysis, most members of this family have a similar topology consisting of 12 transmembrane domains, probably in ␣-helical conformation, suggesting that they are evolutionarily related (1).The lactose permease of Escherichia coli (LacY) is a paradigm for members of the MFS (2), which utilize free energy from an electrochemical proton gradient to catalyze the energetically uphill transport of D-galactopyranosides across the cell membrane or vice versa (3). Typical of the MFS, LacY contains 12 transmembrane helices connected by hydrophilic loops, with both the N and C termini on the cytoplasmic face of the membrane (4 -7), and it is functionally and structurally a monomer (7-9).LacY is selective for disaccharides containing a D-galactopyranosyl ring as well as D-galactose (10 -12) but does not interact with D-glucopyranosides or D-glucose (12)(13)(14). Therefore, the substrate specificity of LacY is directed toward the galactopyranosyl ring of substrate and, most importantly, toward the C-4 OH of the galactopyranosyl ring. Galactose is the most specific substrate of LacY but has a very low affinity (K d(app) ϳ 30 mM) (12), and substitutions at the anomeric position can markedly increase affinity up to 3 orders of magnitude by nonspecific interactions (15).An important advance with regard to unraveling the mechanism of LacY has been the solution of the x-ray structure of the inward facing conformation of a LacY mut...

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

“…The irreplaceable residue E126 is in proximity of R144 and may interact with the O 4 , O 5 , or O 6 atoms of the galactopyranosyl ring via water molecules. A direct interaction between R144 and E126 is not observed in the ligand-bound structure, consistent with the idea that the salt bridge is absent in the presence of ligand (22,23). A hydrophobic interaction between the bottom of the galactopyranosyl ring and the indole ring of Trp151 is observed and supported by phosphorescence studies (24).…”

Electrophysiological characterization of LacY

“…In both mutants, a rapid but weakly electrogenic reaction with similar magnitude and kinetic properties is seen, which agrees with our notion that periplasmic H ϩ binding is not responsible for the charge translocation observed, and that the rapid initial charge displacement of minor electrogenicity is associated with sugar binding. Possibly, the rapid conformational transition after sugar binding (35) leads to rearrangement of charged residues within LacY (3,40), which may account for this phenomenon.…”

Analysis of Glucose Transporter Topology and Structural Dynamics

“…Peptides were eluted using one of two gradients comprising solvent A (5% 1:1 (v/v) acetonitrile:isopropanol in water) and solvent B (1:1 (v/v) acetonitrile:isopropanol). Both solvents contained 0.1% formic acid and 0.01% trifluoroacetic acid (30,31). Supernatant fractions were resolved using 15-min equilibration in 98% solvent A and 2% solvent B followed by raising the percentage of solvent B to 10% over 3 min, to 45% over 37 min, and to 95% over 2 min where it was held for 10 min prior to re-equilibration.…”