THE ROLE OF TRYPTOPHAN-371 AND TRYPTOPHAN-395 IN THE BINDING OF ANTIBIOTICS AND THE TRANSPORT OF SUGARS BY THE D-GALACTOSE-H-COLI( SYMPORT PROTEIN (GALP) FROM ESCHERICHIA)

The interactions between the D-galactose-H+ symporter (GalP) from Esch erichia coli and the inhibitory antibiotics, cytochalasin B and forsko lin, and the substrates, D-galactose and H+, have been investigated fo r 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 r elationship could be assessed. Neither mutation prevented photolabelin g by either [4-H-3]cytochalasin B or by zidophenethyl-amido-7-O-succin yldesacetylforskolin ([I-125]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 cytochala sin B or forskolin. Moreover, both mutations cause a substantial decre ase 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 reduct ions, respectively, Both these mutations change the equilibrium betwee n the putative outward- (T-1) and inward-facing (T-2) conformations, s o that the inward-facing form is more favored. They also stabilize a d ifferent conformational state, ''T-3-antibiotic,'' in which the initia l interactions between the protein and antibiotics are tightened. Over all, this has the effect of compensating for the reduction in affinity for cytochalasin B, so that the respective overall K-d values are 0.7 4- 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 fors kolin. The Trp-371 --> Phe mutation causes a 15-fold reduction in the affinity of the inward-facing site for D-galactose, suggesting that th is residue forms part of the sugar binding site. In contrast, the Trp- 395 --> Phe mutation has no effect upon the affinity of the inward-fac ing site for D-galactose. These effects may be related to the reductio n in galactose-H+ symport activity only in the Trp-371 --> Phe mutant, although it still effects active transport to the same extent as the Trp(395) --> Phe mutant, However, there is a 10-20-fold increase in th e K-m values for energized transport of D-galactose for both mutants.