title: Asparagine 394 in putative helix 11 of the galactose-H+ symport protein (GalP) from Escherichia coli is associated with the internal binding site for cytochalasin B and sugar.
abstract: The galactose-H+ symport protein (GalP) of Escherichia coli is very similar to the human glucose transport protein, GLUT1, and both contain a highly conserved Asn residue in predicted helix 11 that is different in a cytochalasin B-resistant member of this sugar transport family (XylE). The role of the Asn394 residue (which is predicted to be in putative trans-membrane alpha-helix 11) in the structure/activity relationship of the D-galactose-H+ symporter (GalP) was therefore assessed by measuring the interaction of sugar substrates and the inhibitory antibiotics, cytochalasin B, and forskolin with the wild-type and Asn394 --> Gln mutant proteins. Steady-state fluorescence quenching experiments show that the mutant protein binds cytochalasin B with a Kd 37-53-fold higher than the wild type. This low affinity binding was not detected with equilibrium binding or photolabeling experiments. In contrast, the mutant protein binds forskolin with a Kd similar to that of the wild type and is photolabeled by 3-125I-4-azido-phenethylamido-7-O-succinyl-desacetyl-forskolin. The mutant protein displays an increased amount of steady-state fluorescence quenching with the binding of forskolin, suggesting that the substitution of the Asn residue has altered the environment of a tryptophan, probably Trp395, in a conformationally active region of the protein. Time-resolved fluorescence measurements on the mutant protein provided association and dissociation rate constants (k2 and k-2), describing the initial interaction of cytochalasin B to the inward-facing binding site (Ti), that are decreased (9-fold) and increased (4.9-fold) compared with the wild type. This yielded a dissociation constant (K2) for cytochalasin B to the inward-facing binding site 44-fold higher than that of the wild type. The binding of forskolin gave values for k2 and k-2 3.9- and 3.6-fold lower, respectively, yielding a K2 value for Ti similar to that of the wild type. The low overall affinity (high Kd) of the mutant protein for cytochalasin B is due mainly to a disruption in binding to the Ti conformation. It is proposed that Asn394 forms either a direct binding interaction with cytochalasin B or is part of the immediate environment of the binding site and that Asn394 is in the immediate environment, but not part, of the forskolin binding site. The ability of the mutant protein to catalyze energized transport is only mildly impaired with 4.8- and 2.1-fold reduction in Vmax/Km values for D-galactose and D-glucose, respectively. In stark contrast, the overall Kd describing binding of D-galactose and D-glucose to the inward-facing conformation of the mutant and their subsequent translocation across the membrane is substantially increased (64-fold for D-galactose and 163.3-fold for D-glucose). These data indicate that Asn394 is associated with both the cytochalasin B and internal sugar binding sites. This conclusion is also supported by data showing that the sugar specificity of the mutant protein has been altered for D-xylose. This work powerfully illustrates how comparisons of the aligned amino acid sequences of homologous membrane proteins of unknown structure and characterization of their phenotypes can be used to map substrate and ligand binding sites.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom.
authors.forename: T P
authors.initials: TP
authors.lastname: McDonald
authors.forename: A R
authors.initials: AR
authors.lastname: Walmsley
authors.forename: P J
authors.initials: PJ
authors.lastname: Henderson
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D005576:Colforsin / Q000378:metabolism; D003571:Cytochalasin B / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins
chemical_list: D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D005576:Colforsin; D003571:Cytochalasin B; D001216:Asparagine; D005947:Glucose; D005998:Glycine; D005690:Galactose