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.

According to the chemiosmotic principles, secondary active transporters comprise membrane proteins that couple in an obligatory fashion the discharge of an ionic gradient (or that of a solute gradient) to the “uphill” translocation of different solutes in the same direction (symporters or cotransporters) or in the opposite one (antiporters or exchangers) (1). Thermodynamic considerations and a wealth of kinetic, biochemical, and biophysical studies have led to the consensual view that substrate translocation relies on the alternating-access concept (2), stating that at any moment a single binding site in a polar cavity is accessible to only one side of the membrane (see for example, recent reviews and references therein in refs. 3–8). The recent elucidation of the atomic structure of almost a dozen of transporters provides strong support to the validity of the alternating-access concept (see reviews cited above). Finally, the diversity of conformation(s) adopted in the different transporters crystals has yielded insights into the structural basis of the various steps of the symporter cycle. Still, many issues regarding the conformational changes, especially those involved in ligand binding and in the coupling of the ligand binding sites, remain largely unanswered.

In this context, the melibiose permease (MelB) of Escherichia coli, which belongs to the Glycoside-Pentoside-Hexuronide:Cation symporter family (9) (a submember of the major facilitator superfamily, MFS), is a convenient Na⁺ symporter to analyze the molecular and structural basis of the interaction of the coupling ion with the transporter. MelB efficiently couples the uphill transport of α- or β-galactosides to the favorable entry of Na⁺, Li⁺, or H⁺ (H₃O⁺) (10,11). In the past, this property has been extensively exploited to investigate the molecular and structural basis of the ion–MelB interaction and implications in the coupling properties inherent to the symport mechanism (9, 12–16). This strategy led to show, for example, that the three cations compete for the same binding site, that the sugar/ion stoichiometry is 1/1 and that either cosubstrate enhances the affinity for its companion substrate as a manifestation of their coupling (9, 17). MelB models strongly suggest that it is a 12 transmembrane (TM) helices transporter (18–23). Its structural fold (24) (see Fig. 1) is expected to follow that of crystallized transporters of the MFS superfamily (25–27) rather than the one observed in all the Na⁺ symporters so far crystallized, which display a structurally conserved inverted repeat of five TM helices (see reviews cited above). Accordingly, the MelB fold most likely consists in two distinct N-terminal and a C-terminal six-helix bundles, pseudosymmetrically related by an axis running through a central cavity nearly perpendicular to the membrane (24), as other members of the MFS superfamily show (28). As for the above cited symporters, the MelB transport cycling model includes several intermediate steps (12, 30) (Fig. S1). Evidence that conformational changes occur at different stages of the MelB transport cycle has been obtained using biochemical and electrophysiological methods, intrinsic and fluorescence energy transfer spectroscopy, and substrate-induced infrared difference (IR_diff) spectroscopy by attenuated total reflection (ATR) (13, 15, 31–34).

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. 24.

In the past, several mutagenesis studies have shown that four Asp residues (Asp19, Asp55, Asp59, Asp124), located in the putative N-terminal six-helices bundle of MelB, are crucial for Na⁺-dependent affinity increase and transport of melibiose, justifying their assignment as Na⁺ ligands (35–38). Nevertheless, direct evidence showing that mutation of these residues directly interferes with Na⁺ binding is still lacking and the assigned role of these residues remains still tentative.

The aim of this work was to reevaluate the role of these Asp residues in MelB, with special emphasis in their involvement in the Na⁺ and melibiose binding and in their coupling process. To do so we have mostly relied on substrate-induced IR_diff spectroscopy by ATR. In this technique, substrate-induced IR_diff spectra are measured in response to the interaction of the coupling ion with MelB or to the interaction of the sugar with the ion–MelB binary complex (32, 33, 39). The structural information contained in the amide I and amide II regions of the IR_diff spectra provides insight into the conformational changes occurring at these early stages of the symporter reaction, including those related to the coupling process. To aid in the characterization and quantitative interpretation of IR_diff spectra we have applied a spectral correlation analysis. The results from IR_diff spectroscopy have been complemented by intrinsic fluorescence spectroscopy. Our results point to which residues are involved in Na⁺ binding and suggest a sound mechanism for the coupling of the Na⁺ and sugar binding sites. Finally, we used experimentally plausible homology models of MelB for a more detailed interpretation of our results.

Results

In the IR_diff technique the concentrations of substrates in contact with the protein are modulated by a continuous flow of buffers, probing only the sample nearby (∼0.7 μm) to the ATR crystal surface (Fig. 2A and Fig. S2). Given the large and widely distributed number of IR active vibrations in a protein, almost any interaction with the substrate will induce an IR_diff spectrum (40), in contrast to other spectroscopic methods relying on localized probes. Information about protein structural changes is mainly constrained to the 1,700–1,500 cm^-1 region (amide I and II), whereas changes in amino acid side chains are more broadly distributed in frequencies (Fig. 2B). We have demonstrated previously that IR_diff is able to disclose the conformational changes induced by substrates binding (and translocation) in WT MelB (32, 33). Additionally, a mutant without cysteines with a WT phenotype (hereafter C-less) and a mutant with similar substrate-binding properties to WT but defective in sugar translocation (R141C) have been characterized as well by this technique (39).

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).

Here, the same technique was used for detecting and characterizing substrate-induced conformational changes on mutants of the above-mentioned amino acids, derived from C-less MelB. The C-less and mutants were purified, reconstituted in their native E. coli lipids, deposited over an ATR crystal, and placed in contact with a buffer solution (see Materials and Methods and Fig. 2A). From a quantitative comparison of the shape of the structure-sensitive amide I and amide II protein bands of the absorbance spectra, we confirmed that the introduced mutations had a small (for D55C, D59C, and D124C) or insignificant (for D19C and C-less) structural effect on MelB (Fig. S3). This observation reasonably discards that the observed defective substrate-binding phenotypes (see below) could be due to loss of the protein native structure.

Effect of Na⁺ Binding on the Protein Structure.

In the control C-less permease addition of 10 mM Na⁺ to the medium (threefold above the affinity constant) (30) generates a reproducible difference spectrum, formally originated from the substitution of a proton (H₃O⁺) for Na⁺ in the cation-binding site. This difference spectrum includes many discrete spectral bands well above the noise level (Fig. 2B). Peaks in the difference spectrum reflect not only interaction(s) of Na⁺ with the cationic-binding site ligands and associated local structural adjustments, but also induced protein structural changes responsible for the well-established increase in sugar affinity following Na⁺ binding. Even if only few of the arising peaks have been tentatively assigned to given amino acids or to specific secondary structure components (32, 33), the difference spectrum in the protein amide I and II regions offers a powerful fingerprint to globally and quantitatively characterize the structural response of the different mutants to Na⁺ with respect to C-less permease.

To make the quantitative comparison between any mutant and C-less as unbiased as possible, a linear regression analysis encompassing the structure-sensitive 1,710–1,500 cm^-1 region from their difference spectra was applied (see Materials and Methods and Fig. S4 for further details). This global analysis provides two outputs (Fig. 2C). First, the linear correlation parameter, R², quantifies the spectral similarity of a mutant response relative to the C-less, i.e., the percentage of spectral features in common with the control C-less. A high spectral similarity for a mutant is expected to correlate with structural changes in response to the substrate highly similar to those of the C-less. Second, the slope of the linear correlation gives the relative intensity of the spectral features in common with the C-less intensity. A relative intensity lower than 100% for any given mutant implies either a reduced affinity for the added substrate or smaller structural changes in response to substrate binding than for the C-less.

As seen in Fig. 2B, D55C and D59C did not show any clear peak assignable to the protein in their difference spectra (flat signal below ∼1,720 cm^-1). Even after rising the Na⁺ concentration up to 50 mM the spectral response was less than 4% of that observed in the C-less at 10 mM (Fig. 2C), suggesting a major reduction in the affinity constant for Na⁺. The inability of these mutants to bind Na⁺ is further confirmed by a similar response of a negative control containing E. coli lipids without MelB (Fig. 2b), and also by their insignificant spectral similarity with respect the C-less (Fig. 2C). These results confirm that Asp55 and Asp59 are essential side chains for Na⁺ binding. In contrast, D19C displayed an almost WT- or C-less-type signal in terms of intensity and similarity (Fig. 2 B and C), meaning that the previous suggestion that Asp19 is involved in Na⁺ binding (9) is not correct.

The behavior of D124C deserves some special comments. This mutant displays a Na⁺-induced IR_diff spectrum, indicating that it retains the ability to bind Na⁺ (Fig. 2B), implying that Asp124 is not essential for Na⁺ binding to MelB. However, the resulting difference spectrum shows a moderate similarity (∼50%) and a reduced intensity (∼15%) with respect to the C-less difference spectrum (Fig. 2C). The moderate similarity suggests that the Na⁺-induced structural changes of D124C may be less complete that those occurring in C-less. This conclusion is supported by the fact that D124C lacks some intense peaks present in C-less (e.g., at 1,640 and at 1,575 cm^-1, Fig. 2B), whereas all peaks in D124C IR_diff spectrum are also seen in C-less. On the other hand, the markedly smaller intensity of the difference spectrum of D124C appears not to be completely due to a reduced affinity for Na⁺. Increasing the Na⁺ concentration from 10 to 50 mM increased the D124C signal 1.9-fold, suggesting an increase of the Na⁺-affinity constant from 3 to ∼15 mM as a consequence of Asp124 mutation. Such a reduction of affinity could only account for a twofold reduction in the intensity of the IR_diff spectrum with respect the C-less, whereas a sevenfold reduction was observed instead. Consequently, not only the structural changes induced by Na⁺ in D124C are less complete than in C-less, but are also of smaller amplitude, i.e., Asp124 is required for full and native-like structural protein changes in response to Na⁺ binding.

To further complete the characterization of the studied mutants, we performed a comparison of the similitude of the Na⁺-induced IR_diff spectra across all mutants, collected in a correlation matrix (Fig. S4C) used to construct a dendrogram (conceptually similar to a phylogenetic tree) (Fig. 2D). The responses of the D55C and D59C mutants and of the lipid sample appear unclustered, as expected for samples without any specific interaction with Na⁺. In contrast, D19C and D124C are clustered together with the C-less, reflecting that they bind Na⁺. However D124C is the most distant among them, highlighting the above-commented notion that in D124C the interaction with Na⁺ triggers structural changes that differ from C-less and D19C.

Binding of Melibiose in the Absence of Na⁺.

We next explored how each mutation affects the MelB ability to bind melibiose in the absence of Na⁺, i.e., when the only possible coupling ion is H⁺. In these experiments the sugar was added at a concentration of 50 mM (Fig. S2), a value close to the half-saturating concentration of C-less for melibiose (30). Because protons are present in the medium, substrate translocation becomes possible and it may contribute also to the difference spectra. We should note, however, that this contribution is not the dominant one, because R141C that binds but does not translocate the cosubstrates (41) shows only a modest modification of the sugar-induced IR_diff signal compared to that of the functional C-less (39).

Fig. 3A shows that mutants D55C and D59C gave rise to a melibiose-induced IR_diff spectra reasonably similar to that of the C-less (∼70%), even if the decreased intensity in the case of D59C (∼35%) may suggest a ∼4-5-fold reduction of the sugar affinity (Fig. 3B). After a complete spectral similarity comparison (Fig. S5C) both the IR_diff spectra of D55C and D59C mutants appear clustered with that of the C-less (Fig. 3C), reflecting a similar melibiose binding phenotype in terms of protein structural changes. This result provides additional evidence that melibiose binds to these two mutants in a native-like way.

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).

In contrast, D19C and D124C gave rise to small and rather featureless melibiose-induced difference spectra (Fig. 3A), with nearly negligible intensity (∼5%) and low similarity (< 15%) with respect to C-less (Fig. 3B). Even if low, the melibiose-induced responses of D19C and D124C are reproducible and similar among them (Fig. 3C). Also, they are clearly distinct and distant from the difference spectrum observed in a sample containing only E. coli lipids (Fig. 3 A and C). Although their very low intensity may suggest a large decrease in the affinity for melibiose, their low similarity with the C-less (Fig. 3B) suggests a far more dramatic effect of these mutations. In this respect, note the striking few details of the IR_diff spectra for D19C and D124C (mostly limited to one positive and negative band in the amide I region), implying that although melibiose does bind to these two mutants, it interacts poorly with the protein.

In summary, the data indicate that the D19C and D124C mutants display no or very limited conformational changes in response to addition of melibiose, whereas D55C and D59C retain a C-less-like sugar-induced structural variations.

Binding of Melibiose in the Presence of Na⁺.

The shape of the melibiose-induced IR_diff spectra of mutants (Fig. 3 D and F) were not affected by the presence of 50 mM Na⁺ in the medium, in contrast to the C-less at 10 mM Na⁺ (compare Fig. 3A and D; note the different scale in these figures). Moreover, the presence of Na⁺ did not enhance the intensity of the IR_diff spectrum induced by melibiose, as observed in the C-less (Fig. 3E). Therefore, the coupling between the cation- and sugar-binding sites, reflected in the synergistic enhancement of the melibiose-induced IR_diff spectrum by Na⁺, is not functional in any of the mutants. Although this behavior can be (partially) accounted for their impaired Na⁺ (D55C, D59C) and melibiose (D19C, D124C) binding phenotypes, it is possible that at least some of these residues are also required for a functional coupling mechanism.

The correlation matrix and the constructed dendrogram (Fig. S6 C and D) confirm that none of the mutants display a native-like response to melibiose in the presence of Na⁺. Both the spectral responses of mutants with retained melibiose binding (D55C and D59C) and sodium binding (D19C and D124C) were closer among them than to the C-less, in agreement with the notion of an impaired substrate coupling.

Fluorescence Spectroscopy of Mutants.

As a result of the substrate coupling, Trp fluorescence intensity increases upon binding of Na⁺ to MelB WT or C-less in the presence of melibiose, reflecting conformational changes responsible for the Na⁺-induced increase in the affinity for the melibiose (13). None of the mutants studied here showed any significant intensity variation upon incubation with melibiose or with Na⁺ (in the presence of melibiose) (Fig. S7). As indicated previously, comparison of the mutant’s FTIR absorbance spectra discarded overall structural alteration in the protein caused by the mutations. Therefore, we can conclude that the structural changes responsible for the increase in the melibiose affinity upon Na⁺ binding and affecting Trp residues environment, are lacking in the mutants. In agreement with the IR_diff results, the fluorescence results confirm that none of the studied mutants preserves a coupling mechanism between binding of Na⁺ and the sugar. Depending on the particular mutant, this behavior may be caused by different defects (lack of sodium or of sugar binding, or impairment of coupling).

Discussion

On the MelB Na⁺ Ligands.

Taking into account the high sensitivity of infrared spectroscopy, any change in the protein structure or in the protonation state of side chains should be detected in the substrate-induced IR_diff spectra. Therefore, we can conclude that (i) Asp55 and Asp59 are essential ligands to Na⁺ because neither D55C nor D59C exhibit any structural variations upon incubation with the Na⁺ coupling ion. This conclusion is consistent with previous functional results. D59C does not show any transport coupled to H⁺ or Na⁺, implicating this side chain in cation and/or melibiose binding (35, 42, 43). D55C, which still shows measurable transport with H⁺, has selectively lost the capacity to cotransport the sugar with Na⁺, suggesting an implication of Asp55 in Na⁺ binding (37, 38). (ii) Asp19 does not interact with Na⁺, because D19C displays a Na⁺-induced difference spectrum nearly identical to that of C-less. Asp19 was formerly suggested as a ligand to Na⁺ due to its impaired melibiose transport (9), which we can now explain from its involvement as a melibiose ligand. (iii) Most probably, Asp124 interacts with Na⁺ because D124C gives rise to a difference spectrum of only 15% intensity and ∼50% similarity when compared to C-less. An attractive interpretation is that Asp124 is a conditional ligand, establishing an interaction with Na⁺ only when the ion is already bound to Asp55 and Asp59. Although not essential for Na⁺ binding, this interaction would be favorable, increasing the affinity for Na⁺. But more importantly, it would presumably lead Asp124 to approach the bound Na⁺, driving by these means part of the observed Na⁺-induced conformational changes, the ones lacking in D124C. Such hypothesis is further developed below in view of molecular models for MelB.

Side Chains Interacting with Melibiose.

Our study suggests which side chains may serve as melibiose ligands. D19C and D124C showed only small and rather featureless difference spectra upon melibiose incubation in the presence of H⁺ or Na⁺. Therefore, Asp19 and Asp124 are most likely ligands to melibiose, which is in agreement with the absence of transport for D19C and D124C (36, 42, 44). But in spite of their low intensity, the melibiose-induced difference spectra of these two mutants showed a reproducible and similar shape (Fig. 3), implying some interaction of melibiose with MelB. It may be possible that in D19C and D124C the affinity of the active binding site for melibiose drops to the point that melibiose binding can only induce marginal structural changes. Alternatively, the observed faint spectroscopic changes could originate from the interaction of melibiose with a secondary low-affinity binding site as described for the LeuTAa transporter (45).

Concerning Asp55 and Asp59, the melibiose-induced IR_diff spectra of their mutants are of significant intensity. Therefore, we can conclude that Asp55 and Asp59 are not essential residues for melibiose binding. When the coupling ion is H⁺, the decreased signal of D55C and D59C as compared to C-less may be due either to the contribution of these residues to conform the right binding environment for the sugar, to defective H⁺ binding in these mutants, or both. In the presence of Na⁺, the spectra are similar to those obtained in the presence of H⁺, in shape and intensity, showing that there is absence of Na⁺ binding in these two mutants even after melibiose is bound. The lack of Na⁺ binding makes it unfeasible to obtain information about the role of Asp55 or Asp59 in melibiose interaction when Na⁺ is the coupling cation.

Our conclusions agree well with functional properties previously described. An example is provided by binding studies of D55C and D59C. Using the high affinity melibiose analog p-nitrophenyl-alpha-D-galactopyranoside, an affinity around 70% of C-less in the presence of H⁺ and between 15% and 25% in the presence of Na⁺ where obtained (36). On the other hand, substitution of Asp55 with Cys does not drastically modify the H⁺-driven sugar translocation properties of MelB (37). These results are in keeping with the difference spectra showing high percentage of similarity and intensity (Fig. 3 A and B).

Theoretical Models for MelB.

Because no atomic structure with appropriate resolution for MelB is yet available, we relied on theoretical models to evaluate the side chains that shape the binding sites, as well as possible coupling mechanisms between both sites. A recently published model is shown in Figs. 1 and 4A, corresponding to an inward-facing conformation (24). In this model, Asp55 and Asp59 are well positioned to directly participate to the coordination of the Na⁺ coupling ion, a logical outcome because they are included in the same TM helix and are separated by four amino acids. In addition, Asp19 is at more than 14 Å from the presumed cationic binding site, and unlikely to be directly involved in Na⁺ binding. These structural features of the model are consistent with our experimental data. However, Asp124 is at a distance of about 12 Å from the possible Na⁺ site (Fig. 4A), leading the authors to propose (24) that this acidic residue could not be directly interacting with Na⁺. This suggestion makes it less straightforward to explain the significant decrease of the Na⁺-induced structural changes we observed for D124C.

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⁺.

Of help to resolve this difficulty in the interpretation of our experimental data is a homology 3D structure model for MelB (hereafter called I-Tasser MelB model) obtained using the I-Tasser server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) (46). The server took the glycerol-3-phosphate transporter (26) as the preferred template for the modeling and the lactose permease (25) as an additional template, both belonging to the MFS family (see Materials and Methods). One of the five structures with best scores is shown in Fig. S8. In view of the choice of the templates, it is not surprising that the overall architecture of the I-Tasser model bears strong resemblance with that of members of the MFS family. More important for this discussion is its similarity to the Yousef-Guan’s model. In agreement with previous superimposition of the LacY and GlpT templates used here to predict the MelB structure (28), there is a denser helix packing in the I-Tasser MelB model (GlpT-like) than that in the Yousef-Guan’s model (LacY-like). At a closer view, Fig. 4 shows the midregion of the four N-terminal helices (TM1-TM4) with a Na⁺ manually docked at the level of the putative Na⁺ binding site in the Yousef-Guan’s and the I-Tasser models, respectively. Both models agree to suggest a location of Asp19 at more than 9 Å from the Asp55/Asp59 dyad and therefore do not favor a direct interaction with the bound cation. At variance with the Yousef-Guan’s model, however, Asp124 is closer to the hypothetical Na⁺ site in our model. In view of this shorter distance, it is conceivable that after Na⁺ binding, Asp124 could directly interact with the Na⁺ ion by means of a conformational change, which could involve a small change in the relative tilt (and/or producing a rotation) of TM2 and TM4 (47, 48). Such a Na⁺-induced tilt (or rotation) of TM4 in particular, may have important impact on the Na⁺-sugar coupling process, as it has been previously suggested that TM4 may act as a hinge between the Na⁺- and sugar-binding sites (49). Mutation of Asp124 would inhibit these additional structural changes, explaining the reduced Na⁺-induced spectral changes observed in D124C in spite of a retained Na⁺ binding.

We should cautiously note that the MelB models presented here were obtained from distantly homologous templates having only about 15% sequence homology with MelB. However, structural studies of members of the major facilitator superfamily evidenced that they share structural homology, the structural fold being more conserved than sequence (50,51). Therefore, MelB is expected to follow a similar structural fold as the two templates used (24). In any case, the models in Fig. 4 are not claimed to accurately predict the actual conformation of the Na⁺-binding site of MelB, but to suggest reasonable locations of Asp side chains that have been implicated in Na⁺ binding.

Concluding Remarks.

In summary, we combined site directed mutagenesis and substrate-induced IR_diff spectroscopy to reevaluate the implication of some key residues of MelB in substrate binding and in the coupling of the binding sites. This approach should prove useful for the identification and/or reevaluation of important residues in many other membrane transporters, with the added benefit of maintaining the protein in the native lipidic milieu. We foresee that the structural sensitivity of IR spectroscopy, only partially exploited in the present work, will make possible linking the functional and the structural properties of membrane transporters, a task otherwise difficult to be addressed by the incompatible sample requirements of most functional (e.g., biochemical) and structural (e.g., crystallographic) methods.

Materials and Methods

The details of protein expression, purification, and reconstitution of MelB mutants; sample preparation and FTIR spectra acquisition and data treatment; fluorescence spectroscopy; and MelB modeling, are described in the SI Text. In short, MelB mutants harboring a His tag were expressed in E. coli, solubilized in detergent, and purified by affinity chromatography. The solubilized protein was reconstituted into E. coli total lipid liposomes. IR_diff spectra were obtained with a FTS6000 Bio-Rad spectrometer accumulating a total of > 25,000 scans for every difference spectrum. Fluorescence measurements were performed using a UV-visible QuantaMaster™ spectrofluorimeter. MelB modeling was done with the I-Tasser server (46).