Structural insights into the activation mechanism of melibiose permease by sodium binding

Abstract

The melibiose carrier from Escherichia coli (MelB) couples the accumulation of the disaccharide melibiose to the downhill entry of H(+), Na(+), or Li(+). In this work, substrate-induced FTIR difference spectroscopy was used in combination with fluorescence spectroscopy to quantitatively compare the conformational properties of MelB mutants, implicated previously in sodium binding, with those of a fully functional Cys-less MelB permease. The results first suggest that Asp55 and Asp59 are essential ligands for Na(+) binding. Secondly, though Asp124 is not essential for Na(+) binding, this acidic residue may play a critical role, possibly by its interaction with the bound cation, in the full Na(+)-induced conformational changes required for efficient coupling between the ion- and sugar-binding sites; this residue may also be a sugar ligand. Thirdly, Asp19 does not participate in Na(+) binding but it is a melibiose ligand. The location of these residues in two independent threading models of MelB is consistent with their proposed role.

Fig. 1.

Yousef-Guan’s model of the MelB structure obtained by threading through a crystal structure of the lactose permease of E. coli (24). (A) Two views of the solvent excluded surface of the model including helices I, II, and IV as a reference. (B) A cartoon representation of the protein backbone, with helices I, II, and IV highlighted. The putative Na⁺ ligands as proposed in Poolman et al. (9) are shown in stick representation (Asp19, Asp55, Asp59, and Asp124). The figure was produced from the coordinates provided in ref. .

Fig. 2.

Revealing MelB residues participating to Na⁺ binding. (A) MelB reconstituted in E. coli lipids in contact with Na⁺-free buffer (100 mM KCl, 20 mM MES, pH 6.6) is probed by the IR evanescent electromagnetic field generated in the ATR crystal–sample interface. (B) Na⁺-induced IR difference spectra at 4 cm^-1 resolution of MelB C-less and C-less mutants of candidate residues. Difference spectra were obtained by replacing Na⁺-free buffer by a buffer media containing Na⁺ at the concentration indicated on the left-hand side. The buffer exchange protocol and data acquisition scheme are shown in Fig. S2 A and B. All the difference spectra (here and in Fig. 3) were normalized to the amount of probed protein (Fig. S3). The average difference spectrum from three or more (D19C, D124C, and D59C) or two (C-less and D55C) independent experiments is shown, with each experiment representing here and in Fig. 3 the average from at least 25 repetitions conducted on each sample. A sample containing only E. coli lipids was used as negative control. For visualization purposes, the D124C difference spectrum is also shown after multiplication by 3.5. (C) Spectral similarity and intensity of the Na⁺-induced IR difference spectra in the mutants compared to the C-less (see Materials and Methods and Fig. S4). The error bar corresponds to one standard error of the mean. (D) Dendrogram clustering samples according to their spectral similitude in response to Na⁺ (see Materials and Methods and Fig. S4C).

Fig. 3.

Involvement of the studied MelB residues in the melibiose binding and the Na⁺-melibiose coupling. (A) IR difference spectra at 4 cm^-1 resolution of MelB C-less and site-point mutants of the C-less induced by 50 mM melibiose in Na⁺ free media. The buffer exchange protocol and data acquisition scheme are shown in Fig. S2C. The average of three or more (C-less, D19C, D124C, and D59C) or two (D55C) independent experiments is shown. An experiment with only E. coli lipids (without protein) is included as negative control. (B) Comparison of the spectral similarity and intensity of the melibiose-induced IR difference spectra of the C-less mutants with that of the C-less (see Materials and Methods and Fig. S5). Error bars correspond to one standard error of the mean. (C) Dendrogram clustering samples according to their spectral similitude in response to melibiose (see Materials and Methods and Fig. S5C). (D) Melibiose-induced IR difference spectra at 4 cm^-1 resolution of MelB C-less (10 mM melibiose) and several C-less mutants (50 mM melibiose) in the presence of 10 mM Na⁺ (see Fig. S2 D and E). The average difference spectrum from three (D19C) or two (C-less, D124C, D55C, and D59C) independent experiments is shown. An experiment without protein (only E. coli lipids) is included as a negative control. (E) Effect of the presence of Na⁺ in the intensity of the difference spectra induced by melibiose. An increase on the intensity induced by Na⁺ is a signature of a preserved Na⁺-melibiose coupling. (F) Comparison of the spectral similarity and intensity of the melibiose-induced IR difference spectra in the presence of Na⁺ with respect to the C-less (see Materials and Methods and Fig. S6).

Fig. 4.

Working models of ligands for the cation in the putative Na⁺ binding site according to the models of (A) Yousef and Guan (24) and (B) for the one constructed from the I-Tasser server (46). According to the experimental results of this work, Asp55 and Asp59 are obligatory ligands to Na⁺. Eventual participation of main-chain oxygens or water molecule(s) has not been considered. Asp19 does not directly interact with Na⁺, a fact substantiated by both models. The I-Tasser model suggests a possible direct interaction of Asp124 with Na⁺.