Résumé
The periplasmic maltose-binding protein (MBP or MalE protein) of
Escherichia coli is an essential element in the transport of maltose and maltodextrins and in the chemotaxis towards these sugars. On the basis of previous results suggesting their possible role in the activity and fluorescence of MBP, we have changed independently to alanine each of the eight tryptophan residues as well as asparagine 294, which is conserved among four periplasmic sugar-binding proteins.
Five of the tryptophan mutations affected activity. In four cases (substitution of Trp62, Trp230, Trp232 and Trp340), there was a decrease in MBP affinity towards maltose correlated with modifications in transport and chemotaxis. According to the present state of the 2.3A˚three-dimensional structure of MBP, all four residues are in the binding site. Residues Trp62 and Trp340 are in the immediate vicinity of the bound substrate and appear to have direct contacts with maltose; this is in agreement with the drastic increases in
K
d values (respectively 67 and 300-fold) upon their substitution by alanine residues. The modest increase in
K
d (12-fold) observed upon mutation of Trp230 would be compatible with the lesser degree of interaction this residue has with the bound substrate and the idea that it plays an indirect role, presumably by keeping other residues involved directly in binding in their proper orientation. Substitution of Trp232 resulted in a small increase in
K
d value (2-fold) in spite of the fact that this residue is the closest to the ligand of the tryptophan residues according to the three-dimensional model. In the fifth case, replacement of Trp158, which is distant from the binding site, strongly reduced the chemotactic response towards maltose without affecting the transport parameters or the sugar-binding activities of the mutant protein. Trp158 may therefore be specifically implicated in the interaction of MBP with the chemotransducer Tar, but this effect is likely to be indirect, since Trp158 is buried in the structure of MBP. Of course, some structural rearrangements could be responsible in part for the effects of these mutations.
The remaining four mutations were silent. The corresponding residues (Trp10, Trp94, Trp129 and Asn294) are all distant from the sugar-binding site on the crystallographic model of MBP, which is in agreement with their lack of effect on binding. In addition, our results show that they play no role in the interactions with the other proteins of the maltose transport (MalF, MalG or MalK) or chemotaxis (Tar) systems.
The five mutations that affected activity resulted also in alterations in the fluorescence changes occurring upon substrate binding (quenching and shift of peak wavelength), whereas the four silent mutations did not alter these parameters.
This work provides the first genetic evidence for a critical role of tryptophan residues in the activities and fluorescence of a binding protein, as well as another example of a residue conserved in a family of proteins and with no apparent functional role. It indicates that, at least in the case of MBP, the effect on the binding activity of mutating a tryptophan residue to alanine depends primarily on its distance from the sugar-binding site. In addition, all our results are compatible with the idea that fluorescence changes accompanying substrate binding reflect, at least in part, some conformational changes of the protein important for function.