Insights into the Inhibitory Mechanisms of the Regulatory Protein IIA^Glc on Melibiose Permease Activity

Background: The phosphotransfer protein IIA^Glc plays a key role in the regulation of carbohydrate metabolism.

Results: ITC measurements show that IIA^Glc binds to melibiose permease at a stoichiometry of unity and inhibits sugar binding affinity and conformational entropy.

Conclusion: IIA^Glc inhibits MelB by restraining its conformational change.

Significance: IIA^Glc is a useful tool for structure-function studies of its regulated permeases.

Abstract

The phosphotransfer protein IIA^Glc of the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system plays a key role in the regulation of carbohydrate metabolism. Melibiose permease (MelB) is one among several permeases subject to IIA^Glc regulation. The regulatory mechanisms are poorly understood; in addition, thermodynamic features of IIA^Glc binding to other proteins are also unknown. Applying isothermal titration calorimetry and amine-specific cross-linking, we show that IIA^Glc directly binds to MelB of Salmonella typhimurium (MelB_St) and Escherichia coli MelB (MelB_Ec) at a stoichiometry of unity in the absence or presence of melibiose. The dissociation constant values are 3–10 μm for MelB_St and 25 μm for MelB_Ec. All of the binding is solely driven by favorable enthalpy forces. IIA^Glc binding to MelB_St in the absence or presence of melibiose yields a large negative heat capacity change; in addition, the conformational entropy is constrained upon the binding. We further found that the IIA^Glc-bound MelB_St exhibits a decreased binding affinity for melibiose or nitrophenyl-α-galactoside. It is believed that sugar binding to the permease is involved in an induced fit mechanism, and the transport process requires conformational cycling between different states. Thus, the thermodynamic data are consistent with the interpretation that IIA^Glc inhibits the induced fit process and restricts the conformational dynamics of MelB_St.

Introduction

Sugar transport is an important process for all living organisms. To secure the energy supply, bacterial cells usually contain multiple sugar transport systems. Melibiose permease (MelB),² which catalyzes electrogenic symport of galactoside with Na⁺, Li⁺, or H⁺ (1–6), is one of the bacterial sugar transporters. MelB is encoded by the mel operon, which requires transcriptional activation induced by melibiose, as well as a global transcriptional activator (the cAMP-CAP (catabolite activator protein) complex) (7, 8). In certain bacteria, such as Escherichia and Salmonella, the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions, which promotes preferential utilization of glucose (9–11). The phosphotransfer protein IIA^Glc plays a key role in the regulation of carbohydrate metabolism. It regulates the transcription level of various operons encoding non-PTS permeases (12), such as the mel and lac operons, by modulating the level of cAMP. Unphosphorylated IIA^Glc regulates the activity of various non-PTS transporters (9, 11, 13–16). In addition, as a central signaling molecule, it also binds to many other proteins, such as adenylate cyclase (17) and glycerol kinase (18). Recently, it has been reported that IIA^Glc also binds to a carbon storage regulator and affects Vibrio cholerae biofilm formation (19). It is interesting that a single rigid protein is able to bind to various families of soluble proteins as well as membrane transporters. As demonstrated by x-ray crystallography, IIA^Glc binds to the ATPase subunit of maltose permease, preventing structural rearrangements necessary for ATP hydrolysis (16). In the H⁺-coupled lactose permease (LacY), it has been reported that the binding of IIA^Glc requires the presence of sugar (13, 20, 21), but the reported stoichiometry number is not convincing.

In the Na⁺-coupled MelB of Salmonella typhimurium (MeB_St), it was shown that mutations affect the PTS regulation (22, 23); however, there is, at present, no evidence that IIA^Glc directly binds to MelB (11). In addition, there is no information about the energetics of IIA^Glc binding to other proteins. Recently, we solved the three-dimensional crystal structure of MelB_St and demonstrated that MelB (24, 25), a member of the glycoside-pentoside-hexuronide:cation symporter family (26), belongs to a subgroup of the major facilitator superfamily (MFS) permeases (5, 27–29), like LacY (30, 31). The structures were captured in an outward partially occluded and a partially outward-facing conformation, and suggest a single sugar-binding pocket within the central internal cavity (5, 32–34). The structure provides important mechanistic insights for a major facilitator superfamily permease that catalyzes Na⁺-coupled symport (26, 35, 36). However, it was not clear where the IIA^Glc-binding site is and how IIA^Glc regulates MelB activity.

It is generally believed that these IIA^Glc binding partners have little or no sequence or structural homology with one another (37). It is surprising that the C-terminal tail of MelB_Ec and MelB_St, as well as other MelB orthologues, contains the consensus region ⁴⁴³IQIHLLDK⁴⁵⁰ that has a high sequence similarity to ¹²¹LQLAHLLDR¹²⁹ in MalK (16, 38). Both stretches form a short helical structure (16, 25), and the underlined residues in MalK directly contact with IIA^Glc (16). The crystal structure determination of MelB_St reveals that this motif occupies two different conformations; one is closer to the membrane domain (see also Fig. 2c). The previously characterized MelB_St mutants (D438Y, R441S, or I445N) (38), which are resistant to PTS inhibition, are mapped near or within this motif (see Fig. 2c). Based on this structural information, we agree with the previous postulation (38) that the C-terminal fragment of MelB could be a part of the IIA^Glc-binding sites.

FIGURE 2.

ITC measurements of IIA^Glc binding to MelB_St. The protein IIA^Glc (455 μm) was injected into the sample cell containing MelB_St (50 μm), and the heat changes were recorded by a Nano isothermal titration calorimeter (TA Instruments). Both protein samples were matched in the MelB buffer (20 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10% glycerol, and 0.035% undecyl β-d-maltoside) without (black curves) or with 10 mm melibiose (cyan curves). a, thermograms were recorded at 25 °C in the absence or presence of melibiose. Inset, the thermograms were collected by injecting IIA^Glc into the MelB buffer without or with melibiose and presented at an identical scale. b, cumulative heat changes (ΔQ) are plotted as a function of the molar ratios of IIA^Glc/MelB_St and fitted with a one-site independent binding model. Inset, binding energetics in the absence (lighter colors) or presence of melibiose (darker colors). ΔH and K_a were measured directly; K_d = 1/K_a; ΔG = −RT ln K_a; TΔS = ΔH − ΔG. n, stoichiometry. Error bars, S.E., number of tests = 2 or 3. c, the crystal structures of MelB_St (25) and IIA^Glc (16) are shown on the same scale. The crystal structure of MelB_St reveals two different conformations (Mol-A and Mol-B, where Mol-A and Mol-B mean molecule-A and molecule-B). Most of the cytoplasmic middle loop (in yellow) between the N- (in blue) and C-terminal (in green) domains is missing in Mol-B. The backbone of the C-terminal tail until Leu⁴⁴⁸ or Asp⁴⁴⁹ was resolved in Mol-A or Mol-B, respectively (25). The sequences in the stretch ⁴⁴³IQIHLLDK⁴⁵⁰ in the C-terminal tail, which has high similarity with the IIA^Glc-contacting surface in MalK (16), are colored in cyan. The reported mutants (D438Y, R441S, or I445N), which are resistant to PTS inhibition, are shown as spheres (red, blue, or gray, respectively). PDB, Protein Data Bank. d, cross-linking. The DSP-mediated amine-specific cross-linking reactions in the absence or presence of melibiose were analyzed by SDS-12% PAGE and visualized by silver staining.

In this study, we determined the thermodynamics of the IIA^Glc-MelB interaction, sugar binding to MelB_St, as well as the effect of IIA^Glc on the sugar-MelB interaction using isothermal titration calorimetry (ITC). We observed that IIA^Glc binds to MelB_St in the absence or presence of melibiose, and inhibits the conformational entropy and sugar affinity of the transporter.

MATERIALS AND METHODS

Reagents

The Phos-tag^TM acrylamide (NARD Institute, Ltd.). Dithiobis(succinimidyl propionate) (DSP) was from Thermo Scientific. Nitrophenyl-α-galactoside (α-NPG) and nitrophenyl-α-glucoside (α-NPGlu) were from Sigma-Aldrich.

Gene Cloning of IIA^Glc

The gene encoding IIA^Glc was amplified from the chromosomal DNA of Escherichia coli DW2 strain (5) by PCR (sense primer, 5′-TATATGCTCTTCTAGTATGGGTTTGTTCGATAAACTAAAATC-3′; antisense primer, 5′-TATATAGCTCTTCATGCTCATTACTTCTTAATGCGGATAACCGGAGT-3′), cloned into the T7-based expression vector p7XNH3 with a kanamycin resistance marker by the fragment-exchange cloning method (39). The resultant plasmid contains a 10-His tag sequence at the N terminus with a 9-residue linker (MHHHHHHHHHHLEVLFQGPS), which was verified by DNA sequencing analysis.

Protein Expression and Purification

The overexpression of IIA^Glc was performed in the E. coli T7 express strain (New England Biolabs). The cells were grown in LB containing 0.5% glycerol, 0.2% glucose, and 50 mg/liter kanamycin. The overnight cultures were diluted to 2% with the same medium and shaken at 30 °C. Isopropyl-1-thio-β-d-galactopyranoside at 0.4 mm was added at A₆₀₀ of 0.8, and the incubation was continued for another 4 h. Cells were harvested and suspended in a buffer containing 50 mm NaP_i, pH 7.5, 200 mm NaCl, 5% glycerol, and 0.1% PMSF, and broken by passage through an EmulsiFlex at 10000 p.s.i. The supernatant, after ultracentrifugation at 70.409 × g for 30 min in a Beckman rotor, type 45 Ti at 4 °C, was loaded onto a column containing Talon resin (Clontech) pre-equilibrated with 50 mm NaP_i, pH 7.5, 200 mm NaCl, 5% glycerol, 5 mm imidazole for cobalt affinity chromatography. After washing with the same buffer containing 30 mm imidazole, elution was performed using the same buffer containing 200 mm imidazole; the eluate was concentrated to ∼100 mg/ml using a VIVASPIN 20 (5,000 molecular weight cut-off polyethersulfone, Millipore) and dialyzed against three changes of 1 liter of dialysis buffer containing 20 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10% glycerol. The protein concentration of IIA^Glc was measured by the Micro BCA protein assay (Pierce Biotechnology, Inc.). The protein samples were flash-frozen in liquid nitrogen and stored at −80 °C. From a 1-liter culture, about 50 mg of highly pure IIA^Glc protein can be obtained routinely. The purified IIA^Glc protein was analyzed with both SDS-14% PAGE and Phos-tag SDS-12% PAGE.

The overexpression and purification of MelB_St and MelB_Ec (40, 41) were carried out as described (25). MelB proteins in the MelB buffer (20 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.035% undecyl β-d-maltoside, and 10% glycerol) were concentrated to 20 mg/ml, flash-frozen in liquid nitrogen, and stored at −80 °C.

Isothermal Titration Calorimetry

ITC measurements were performed in a Nano isothermal titration calorimeter (TA Instruments). Purified MelB (50 μm) in MelB buffer without or with 10 mm melibiose was placed into the sample cell with a reaction volume of 163 μl, and IIA^Glc (455 μm) in the MelB buffer without or with 10 mm melibiose, prepared by dilution from a highly concentrated protein sample, was titrated incrementally into the MelB sample. For measuring sugar binding to MelB, melibiose at 10 or 100 mm was dissolved in the MelB buffer; α-NPG or α-NPGlu at 1 mm was prepared by diluting 200 mm α-NPG or α-NPGlu in dimethyl sulfoxide into the MelB buffer. In this case, the same concentration of dimethyl sulfoxide was added into the sample placed in the sample cell. When testing the IIA^Glc effect, IIA^Glc was preincubated with MelB at a 2 to 1 ratio for 1 h.

Titrations were performed by injection of titrant with an interval of 250 or 300 s at a constant stirring rate of 250 rpm. By plotting integrated rates of heat change against the molar ratio of IIA^Glc/MelB or sugar/MelB, the binding stoichiometry number (n), the association constant (K_a), and the enthalpy change (ΔH) are directly determined by fitting the data using the one-site independent binding model provided by the instrument. The dissociation constant (K_d) and the entropy change (ΔS) are obtained by calculation using the equation of ΔG = −RT ln K_a and TΔG = ΔH − ΔG, where ΔG is free energy change, R is the Faraday constant, and T is absolute temperature. Parameterization of ΔS was calculated as described previously (42). Total ΔS = ΔS_solv + ΔS_mix + ΔS_conf. Mixing entropy change (ΔS_mix) = R ln (1/55.5) = −33 J/mol/K. Solvent entropy change (ΔS_solv) = ΔC_p ln (298.15/385.15), where ΔC_p is the heat capacity change. Conformational entropy change ΔS_conf = ΔS − ΔS_mix − ΔS_solv.

Protein Cross-linking

The amine-reactive cross-linking reagent DSP was used for the cross-linking reaction between MelB_St and IIA^Glc. Briefly, 1 μg of MelB_St (1.88 μm) in the absence or presence of 10 mm melibiose and IIA^Glc (4.8 μm) in 20 mm HEPES, pH 7.6, 50 mm NaCl, and 0.035% undecyl β-d-maltoside were preincubated for 15 min, and the cross-linking reaction was carried out by incubating with 200 μm DSP for 15 min at room temperature and stopped by the addition of 100 mm Tris-HCl. The reaction samples were analyzed with SDS-12% PAGE and visualized by silver staining.

RESULTS

IIA^Glc Binding to MelB_St in the Absence or Presence of Melibiose

IIA^Glc was purified to homogeneity from the E. coli T7 express strain (Fig. 1), and the Phos-tag SDS-PAGE analysis indicates that the affinity-purified recombinant IIA^Glc protein is unphosphorylated (Fig. 1, right). By titrating IIA^Glc into a MelB_St sample at 25 °C, ITC measurements show exothermic binding in the absence or presence of melibiose (Fig. 2a). No detectable changes were observed when injecting IIA^Glc into the buffer (Fig. 2a, inset) or buffer to MelB_St (data not shown). The data fitting (Fig. 2b) suggests dissociation constant (K_d) values of 3.62 or 10.15 μm (Table 1) in the absence or presence of melibiose, respectively. The protein-protein interaction under both conditions is solely driven by favorable enthalpy change (ΔH) and opposed by negative entropy change (TΔS) (Fig. 2b, inset; Table 1). The results indicate that polar or hydrophilic interactions are the major forces governing IIA^Glc binding and that the charged or polar residues in the proposed IIA^Glc-binding site in the C-terminal tail may contribute to the enthalpy forces (Fig. 2c).

FIGURE 1.

SDS-PAGE and Phos-tag SDS-PAGE analyses. IIA^Glc was overexpressed in the presence of glucose and purified by cobalt affinity chromatography. Each lane on the SDS-14% PAGE and Phos-tag SDS-12% PAGE was loaded with 1 μg of protein and stained with silver nitrate. The Phos-tag SDS-12% PAGE contains 20 μm of Phos-tag.

TABLE 1.

Energetics of IIA^Glc binding to MelB_St at 25 °C

Data are presented in Fig. 2.

	Ka	Kd	ΔG	ΔH	TΔS	ΔS	na
	mol−1	μm	kJ/mol	kJ/mol	kJ/mol	J/mol/K
No sugar	290,433 (436,09b)	3.62 (0.59)	−31.13 (0.39)	−48.74 (2.73)	−17.61 (2.42)	−59.10 (8.12)	0.98 (0.10)
Melibiosec	104,415 (24,785)	10.15 (0.80)	−28.58 (0.60)	−37.91 (1.52)	−9.33 (0.92)	−31.32 (3.09)	0.78 (0.06)

^a Stoichiometry of IIA^Glc versus MelB_St.

^b S.E., number of tests = 2–3.

^c Melibiose at 10 mm was pre-equilibrated with both proteins.

When melibiose is preincubated with MelB_St (Fig. 2a, Table 1), the ΔS becomes less unfavorable. The measured stoichiometry number (n) without melibiose is 0.98. In the presence of melibiose, the n number is about 0.78; however, at 20 °C, it is 1.1 (Table 2). It is noteworthy that asymmetric peaks appear at the beginning of the titration in the absence of sugar (Fig. 2a).

TABLE 2.

Energetics of IIA^Glc binding to MelB_Ec at 20 °C

Data are presented in Fig. 3.

	Ka	Kd	ΔG	ΔH	TΔS	ΔS	na
	mol−1	μm	kJ/mol	kJ/mol	kJ/mol	J/mol/K
No sugar	39,195 (5355b)	25.75 (3.7)	−25.76 (0.33)	−37.49 (0.25)	−11.73 (0.08)	−40.01 (0.28)	1.22 (0.07)
Melibiosec	39,640 (3320)	25.4 (2.10)	−25.80 (0.20)	−39.23 (0.89)	−13.44 (1.10)	−45.84 (3.75)	1.27 (0.11)

^a Stoichiometry of IIA^Glc versus MelB_Ec.

^b S.E., number of tests = 2.

^c Melibiose at 10 mm was pre-equilibrated with both proteins.

The interaction of IIA^Glc with MelB_St was further tested by amine-specific cross-linking studies. In the absence or presence of melibiose, a band with M_r = ∼62,000 was obtained only in the presence of cross-linking reagents, which corresponds to the cross-linked product containing one MelB_St and one IIA^Glc (Fig. 2d). These data are consistent with the results from the ITC measurements and support the conclusion that the stoichiometry of IIA^Glc to MelB_St is unity in the absence or presence of melibiose.

IIA^Glc binding to E. coli MelB (MelB_Ec) was also examined by ITC. The data reveal thermodynamic features similar to that observed when injecting IIA^Glc into MelB_St, except for the higher K_d value of 25 μm (Fig. 3, Table 2); furthermore, there is no difference in the absence or presence of melibiose. The following studies focused only on IIA^Glc binding to MelB_St.

FIGURE 3.

ITC measurements of IIA^Glc binding to MelB_Ec. IIA^Glc (400 μm) was injected into a 30 μm MelB_Ec solution. Both protein samples were in MelB buffer without (black curves) or with 10 mm melibiose (cyan curves). a, thermograms were recorded at 20 °C. b, ΔQ values are plotted as a function of the molar ratio of IIA^Glc/MelB_Ec and fitted with a one-site independent binding model. The thermodynamic parameters were obtained as described in the legend for Fig. 2 and also presented in Table 2. Error bars, S.E.

Inhibition of Conformational Entropy by IIA^Glc

IIA^Glc binding to MelB_St was further tested in the temperatures ranging from 20 to 35 °C. With an increase in temperature (Table 3, Fig. 4a), ΔH values become more favorable, and ΔS exhibits compensation to ΔH, as indicated by the parallel curves. As a result of the compensation, there is little change in ΔG and K_d with temperature. A linear fitting of ΔH versus temperature reveals the heat capacity change (ΔC_p, the slope) of −1.17 and −1.04 kJ/mol/K in absence or presence of melibiose, respectively. There is no significant difference at either condition.

TABLE 3.

Temperature effect

Data are presented in Fig. 4a.

Temperature (in °C)	No melibiose					10 mm melibiosea
	Kd	ΔG	ΔH	TΔS	n	Kd	ΔG	ΔH	TΔS	n
	μm	kJ/mol	kJ/mol	kJ/mol		μm	kJ/mol	kJ/mol	kJ/mol
20	2.62 (0.62b)	−31.38 (0.57)	−42.88 (2.67)	−11.50 (2.10)	0.97 (0.11)	10.42 (0.80)	−27.98 (0.19)	−30.37 (0.39)	−2.39 (0.56)	1.10 (0.06)
25	3.62 (0.59)	−31.13 (0.39)	−48.74 (2.73)	−17.61 (2.42)	0.98 (0.10)	10.15 (2.41)	−28.58 (0.60)	−37.91 (1.52)	−9.33 (0.92)	0.78 (0.06)
30	3.56 (0.60)	−31.66 (0.43)	−54.43 (3.27)	−22.77 (2.84)	0.96 (0.09)	12.30 (2.08)	−29.36 (0.56)	−40.68 (0.66)	−11.32 (0.14)	0.76 (0.07)
35	5.80 (0.75)	−30.97 (0.37)	−60.43 (0.88)	−29.46 (0.86)	0.94 (0.07)	11.79 (2.15)	−29.15 (0.44)	−46.71 (2.97)	−17.56 (2.75)	0.57 (0.11)

^a Melibiose at 10 mm was pre-equilibrated with both proteins.

^b S.E., number of tests = 2–3.

FIGURE 4.

ΔC_p, ΔS_solv, and ΔS_conf. IIA^Glc binding to MelB_St in the absence (empty symbols or lighter color bars) and presence (filled symbols or darker color bars) of 10 mm melibiose was tested at 20, 25, 30, or 35 °C. a, the ΔG (black), ΔH (blue), and TΔS (green) values were obtained as described in the legend for Fig. 2 and plotted against temperature. The ΔC_p values were obtained by a linear fit of ΔH. b, ΔS_solv and ΔS_conf. Both parameters were obtained as described in legend for the Table 4.

Because the ΔC_p value was determined, the ΔS can be parameterized (42) (Table 4; Fig. 4b), which reveals a large increase in the solvent-entropy change (ΔS_solv) and a large unfavorable conformational entropy change (ΔS_conf). Without sugar, the binding yields a more favorable change in ΔS_solv and a more unfavorable change in ΔS_conf.

TABLE 4.

Parameterization of ΔS

	No sugar	10 mm melibiose
ΔCp a (J/mol/K)	−1167	−1036
ΔSb (J/mol/K)	−59.10	−31.32
ΔSmixc (J/mol/K)	−33.00	−33.00
ΔSsolvd (J/mol/K)	298.75	265.21
ΔSconfe (J/mol/K)	−324.85	−263.54

^a ΔC_p = ΔH/ΔT, the slope from the linear fitting (Fig. 4a).

^b Total ΔS = ΔS_mix + ΔS_solv + ΔS_conf .

^c Mixing entropy change ΔS_mix = R ln (1/55.5) = −33 (J/mol/K).

^d Solvent entropy change ΔS_solv = ΔC_p ln (298.15/385.15).

^e Conformational entropy change ΔS_conf = ΔS − ΔS_mix − ΔS_solv.

Inhibition of Melibiose Affinity by IIA^Glc

We further analyzed the effect of IIA^Glc binding on sugar affinity. As we previously showed (25), the binding of melibiose to the Na⁺-bound MelB_St was detected by ITC measurement at 25 °C; the binding is exothermic with a K_d value of 950 μm (Fig. 5a). The binding is driven by both favorable ΔH and favorable ΔS (Table 5). Strikingly, when injecting the melibiose solution to the MelB_St-IIA^Glc complex at 25 °C (Fig. 5b) or 35 °C (data not shown), the heat changes are smaller than the control. By injecting 10-fold higher melibiose (100 mm), a titration curve after correction for the buffer control shows an endothermic effect (Fig. 5c, inset). As a control, injection of 100 mm sucrose (a non-substrate) does not produce detectable binding signals (Fig. 5d). Although the weak signals were not amenable for fitting, the affinity is apparently inhibited.

FIGURE 5.

IIA^Glc effect on melibiose binding to MelB_St. Melibiose prepared in the MelB buffer was used for the titration at 25 °C. a–d, MelB_St at 80 μm without (a) or with preincubation with 160 μm IIA^Glc (b–d). Inset, ΔQ values are plotted as a function of the molar ratios of IIA^Glc-MelB_Ec and fitted with a one-site independent binding model. The free energies values are presented in Table 5. a, titration of melibiose at 10 mm with MelB_St (red) or MelB buffer (blue). b, titration of melibiose at 10 mm with MelB_St-IIA^Glc complex (red) or MelB buffer (blue). c, titration of melibiose at 100 mm with MelB_St-IIA^Glc complex (red) or MelB buffer (blue). d, titration of sucrose at 100 mm with MelB_St-IIA^Glc complex (red) or MelB buffer (blue).

TABLE 5.

Energetics of sugar binding to MelB_St at 25 °C

The binding data are presented in Figs. 5–6.

Sugars	Proteins	Kd	ΔG	ΔH	TΔS
		μm	kJ/mol	kJ/mol	kJ/mol
Melibiose	MelBSt	950 (12a)	−17.28 (0.30)	−9.27 (0.05)	8.00 (0.24)
α-NPG	MelBSt	15.45 (0.35)	−27.46 (0.05)	−43.25 (0.44)	−15.79 (0.49)
α-NPG	MelBSt-IIAGlc	76.13 (4.52)	−23.51 (0.15)	21.28 (5.03)	44.79 (5.17)

^a S.E., number of tests = 2.

Inhibition of α-NPG Affinity by IIA^Glc

When titrating α-NPG into MelB_St at 25 °C, large exothermic peaks were detected (Fig. 6a, red). As the control for the hydrophobic substrate binding to the detergent-solubilized membrane protein, we tested binding of the α-NPGlu. Under the same conditions, injection of α-NPGlu yields a flat titration curve with small exothermic peaks (Fig. 6a, black). The results strongly support the notion that the NPG titration curve reflects a specific binding to MelB_St, which is consistent with the previous conclusion that MelB recognizes di- and trisaccharides containing the galactosyl moiety or galactose (1, 5, 25, 43). The exothermic binding curve was also fitted using the one-site independent binding model (Fig. 6b). The K_d is 15.45 μm, and energetically, the binding is solely driven by ΔH and opposed by ΔS (Fig. 6b, inset; Table 5), which is different from the melibiose binding, as well as the NPG binding to LacY (44).

FIGURE 6.

IIA^Glc effect on α-NPG binding to MelB_St. a–d, α-NPG and α-NPGlu prepared at 1 mm with 0.5% dimethyl sulfoxide-containing MelB buffer were used for the titration at 25 °C. MelB_St at 80 μm without (a and b) or with preincubation with 160 μm IIA^Glc (c and d). a and c, titration of α-NPG (red) and α-NPGlu (black) with MelB_St or MelB_St-IIA^Glc complex. b and d, ΔQ values collected from the α-NPG binding in panel a or c are plotted as a function of the molar ratios of α-NPG-MelB_St, respectively. Inset, binding energetics as presented in Table 5. The one-site independent binding model was used for data fitting. Error bars, S.E., number of tests = 2.

When titrating α-NPG into the IIA^Glc-MelB_St complex, similar to that observed with melibiose, an endothermic thermogram with small heat changes was observed (Fig. 6c, red). The K_d value increases to 76.13 μm, indicating that IIA^Glc inhibits the α-NPG binding by 5-fold. The binding becomes solely driven by ΔS with compensation of ΔH (Fig. 6d; Table 5).

DISCUSSION

Previous studies reported that the binding of IIA^Glc to LacY requires the presence of sugar substrate (13, 20, 21); in contrast, we show here that IIA^Glc binds to MelB_Ec or MelB_St in the absence or presence of the sugar. The binding to MelB_St has higher affinity than that to MelB_Ec. Without or with melibiose, the titration curves of IIA^Glc to MelB_Ec are similar. When injecting IIA^Glc into MelB_St without sugar, the heat change peaks in the beginning of the thermogram are asymmetric, which indicates that IIA^Glc binding is relatively slow. It is likely that MelB_St occupies several conformational states; not all the conformers can be recognized by IIA^Glc, which causes the slow binding. The results suggest that the conformational transition is slow in the absence of melibiose. It is possible that the lower number of MelB_St molecules available for the IIA^Glc binding in the absence of melibiose or at a low temperature (Table 3) results in an apparently faster decrease of the peak height in the titration. Since MelB_Ec is conformationally more flexible, a faster rate of IIA^Glc binding and no sugar effect are observed. Thus, sugar binding facilitates the conformational dynamics associated with IIA^Glc binding.

Although the IIA^Glc-bound state of MelB and the details of the interaction are not known, two conformations, an outward partially occluded conformation and a partially outward-facing conformation, were determined by crystallography (25); both structures show a close surface at the cytoplasmic side, reflecting its low energy state. Accordingly, it seems reasonable to postulate that the cytosolic IIA^Glc binds between the C-terminal tail and a closed face on the cytoplasmic side of MelB (Fig. 2c).

IIA^Glc binding to MelB is involved in a large favorable ΔS_solv with compensation of a large unfavorable ΔS_conf. Because IIA^Glc is structurally rigid (16, 45), it is likely that the large change in ΔS_solv may mainly result from the conformationally flexible MelB_St, as well as the binding interface. The inhibition of conformational entropy upon the binding implies that IIA^Glc binding prevents the conformational changes of MelB, similar to that proposed by the structural approach in maltose permease (16). The less restrained ΔS_conf is observed in the presence of melibiose, which is probably due to the idea that the conformational entropy was restrained to a certain extent by the sugar binding prior to the IIA^Glc binding.

IIA^Glc effects on MelB_St affinity for sugars were examined with the lower affinity melibiose and the higher affinity ligand α-NPG. The ITC measurements show that IIA^Glc binding affects the sugar binding significantly. It inhibits the sugar binding affinity; on the other hand, it alters the thermodynamic features of sugar binding from an exothermic to an endothermic reaction. Because the affinity of MelB_St for melibiose is at the lower sensitivity boundary of the Nano ITC equipment, the decreased sugar binding affinity by IIA^Glc is out of the detectable range of this method. We confirmed the IIA^Glc inhibition of sugar binding with α-NPG. The ITC measurements reveal that α-NPG binds to MelB_St with >60-fold higher affinity than does melibiose. Different from the entropy-driven melibiose binding, the α-NPG binding is solely driven by ΔH, suggesting that the increased affinity mainly results from polar or hydrophilic interactions between α-NPG and MelB_St. Furthermore, α-NPG binding to MelB_St in the IIA^Glc-bound complex becomes solely driven by ΔS with a 5-fold higher K_d value. Because the sugar binding to the permease is likely involved in an induced fit process as demonstrated in LacY (46, 47), the results could be explained by the observed unfavorable ΔS_conf of the IIA^Glc-MelB_St complex. Thus, IIA^Glc inhibits the induced fit process for sugar binding by restricting the conformational entropy.

Overall, the observed direct interaction of IIA^Glc with MelB_St inhibits the sugar affinity and conformational dynamics of the transporter protein. It is likely that by such a mechanism, unphosphorylated IIA^Glc blocks entry of melibiose, the inducer of the mel operon, so that the cells utilize glucose via the PTS transport system.