Abstract
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.
Highlights
IntroductionThe sugar specificity of galactose-Hϩ symporter (GalP) is very similar to that of the human erythrocyte (GLUT1) and the rat adipocyte (GLUT4) glucose transporters (Barnett et al, 1973; Rees and Holman, 1981; Cairns et al, 1991; Walmsley et al, 1994b)
This phenomenon allowed the kinetics of the binding of cytochalasin B to GLUT1 and galactose-Hϩ symporter (GalP) to be monitored by stopped-flow fluorescence spectroscopy, showing that the mechanisms of binding of cytocha
We have shown previously that the affinity of the GalP protein for D-galactose can be determined by measuring the D-galactose inhibition of the apparent rate of binding of cytochalasin B; this technique provides a measure of the affinity of the inward-facing site for the sugar (Walmsley et al, 1993, 1994a, 1994b)
Summary
The sugar specificity of GalP is very similar to that of the human erythrocyte (GLUT1) and the rat adipocyte (GLUT4) glucose transporters (Barnett et al, 1973; Rees and Holman, 1981; Cairns et al, 1991; Walmsley et al, 1994b). The binding of either cytochalasin B or forskolin induces a quench in the protein fluorescence of both GLUT1 and GalP (Gorga and Lienhard, 1982; Carruthers, 1986; Pawagi and Deber, 1990; Chin et al, 1992; Walmsley et al, 1994; Martin, 1993; Martin et al, 1995). This phenomenon allowed the kinetics of the binding of cytochalasin B to GLUT1 and GalP to be monitored by stopped-flow fluorescence spectroscopy, showing that the mechanisms of binding of cytocha-. All these data identified at least three conformational states of the transport proteins, changes in which were detected by measuring protein fluorescence
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