The role of tryptophans 371 and 395 in the binding of antibiotics and the transport of sugars by the D-galactose-H+ symport protein (GalP) from Escherichia coli.
The interactions between the D-galactose-H+ symporter (GalP) from Escherichia coli and the inhibitory antibiotics, cytochalasin B and forskolin, and the substrates, D-galactose and H+, have been investigated for the wild-type protein and the mutants Trp-371-->Phe and Trp-395-->Phe, so that the roles of these residues in the structure-activity relationship could be assessed. Neither mutation prevented photolabeling by either [4-3H]cytochalasin B or by 3-[125I]iodo-4-azidophenethyl-amido-7-O-succinyldesacetylforskolin ([125I]APS-forskolin). However, measurements of protein fluorescence show that both residues are in structural domains, the conformations of which are perturbed by the binding of cytochalasin B or forskolin. Moreover, both mutations cause a substantial decrease in the affinity of the inward-facing site of the GalP protein for cytochalasin B, 10- and 43-fold, respectively, but have little effect upon the affinity of this site for forskolin, 0.8- and 2.6-fold reductions, respectively. Both these mutations change the equilibrium between the putative outward- (T1) and inward-facing (T2) conformations, so that the inward-facing form is more favored. They also stabilize a different conformational state, "T3-antibiotic," in which the initial interactions between the protein and antibiotics are tightened. Overall, this has the effect of compensating for the reduction in affinity for cytochalasin B, so that the respective overall Kd values are 0.74- and 3.5-fold that of the wild type, while causing a slight increase, 1.5- and 3.2-fold, respectively, in affinity of the mutants for forskolin. The Trp-371-->Phe mutation causes a 15-fold reduction in the affinity of the inward-facing site for D-galactose, suggesting that this residue forms part of the sugar binding site. In contrast, the Trp-395-->Phe mutation has no effect upon the affinity of the inward-facing site for D-galactose. These effects may be related to the reduction in galactose-H+ symport activity only in the Trp-371-->Phe mutant, although it still effects active transport to the same extent as the Trp395-->Phe mutant. However, there is a 10-20-fold increase in the Km values for energized transport of D-galactose for both mutants.
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, United Kingdom.
T P
TP
McDonald
A R
AR
Walmsley
G E
GE
Martin
P J
PJ
Henderson
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000378:metabolism / Q000494:pharmacology; D003571:Cytochalasin B / Q000378:metabolism / Q000494:pharmacology; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism*; D005798:Genes, Bacterial; D007457:Iodine Radioisotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D010649:Phenylalanine; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014316:Tritium; D014364:Tryptophan
D000900:Anti-Bacterial Agents; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D007457:Iodine Radioisotopes; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011994:Recombinant Proteins; C023550:galactose-binding protein; D014316:Tritium; D005576:Colforsin; D003571:Cytochalasin B; D010649:Phenylalanine; D014364:Tryptophan; D005690:Galactose