Evidence for a role of helix IV in connecting cation- and sugar-binding sites of Escherichia coli melibiose permease

TO improve the structural organization model of melibiose permease, we asse ssed the individual contributions of the N-terminal tryptophans to the tran sporter fluorescence variations induced by the binding of cations and beta- configured sugars, by replacement of the six N-terminal tryptophans by phen ylalanines and the study of the signal changes. Only two mutations, W116F l ocated in helix IV and W128F located in the cytoplasmic loop 4-5, impair pe rmease activity. The intrinsic fluorescence spectroscopy analysis of the ot her mutants suggests that W54, located in helix II, W116, and W128 are most ly responsible for the cation-induced fluorescence variations. These trypto phans, W116 and W128, would also be responsible for the beta-galactoside-in duced fluorescence changes observed in the N-terminal domain of the transpo rter. The implication of W116 and W128 in both the cation- and beta-galacto side-induced fluorescence variations led us to investigate in detail the ef fects of their mutations on the functional properties of the permease. The results obtained suggest that the domains harboring the two tryptophans, or the residues themselves, play a critical role in the mechanism of Na+/suga r symport. Taken together, the results presented in this paper and previous results are consistent with a fundamental role of helix IV in connecting c ation- and sugar-binding sites of the melibiose permease.