Asparagine 394 in Putative Helix 11 of the Galactose-H+ Symport Protein (GalP) from Escherichia coli Is Associated with the Internal Binding Site for Cytochalasin B and Sugar*

The galactose-H+ symport protein (GalP) of Escherichia coli is very similar to the human glucose transport protein, GLUT1, and both contain a highly conserved Asn residue in predicted helix 11 that is different in a cytochalasin B-resistant member of this sugar transport family (XylE). The role of the Asn394 residue (which is predicted to be in putative trans-membrane α-helix 11) in the structure/activity relationship of the d-galactose-H+ symporter (GalP) was therefore assessed by measuring the interaction of sugar substrates and the inhibitory antibiotics, cytochalasin B, and forskolin with the wild-type and Asn394 → Gln mutant proteins. Steady-state fluorescence quenching experiments show that the mutant protein binds cytochalasin B with a K d 37–53-fold higher than the wild type. This low affinity binding was not detected with equilibrium binding or photolabeling experiments. In contrast, the mutant protein binds forskolin with aK d similar to that of the wild type and is photolabeled by 3-125I-4-azido-phenethylamido-7-O-succinyl-desacetyl-forskolin. The mutant protein displays an increased amount of steady-state fluorescence quenching with the binding of forskolin, suggesting that the substitution of the Asn residue has altered the environment of a tryptophan, probably Trp395, in a conformationally active region of the protein. Time-resolved fluorescence measurements on the mutant protein provided association and dissociation rate constants (k 2 and k −2), describing the initial interaction of cytochalasin B to the inward-facing binding site (Ti), that are decreased (9-fold) and increased (4.9-fold) compared with the wild type. This yielded a dissociation constant (K 2) for cytochalasin B to the inward-facing binding site 44-fold higher than that of the wild type. The binding of forskolin gave values fork 2 and k −2 3.9- and 3.6-fold lower, respectively, yielding a K 2value for Ti similar to that of the wild type. The low overall affinity (high K d ) of the mutant protein for cytochalasin B is due mainly to a disruption in binding to the Ti conformation. It is proposed that Asn394forms either a direct binding interaction with cytochalasin B or is part of the immediate environment of the binding site and that Asn394 is in the immediate environment, but not part, of the forskolin binding site. The ability of the mutant protein to catalyze energized transport is only mildly impaired with 4.8- and 2.1-fold reduction inV max/K m values ford-galactose and d-glucose, respectively. In stark contrast, the overall K d describing binding of d-galactose and d-glucose to the inward-facing conformation of the mutant and their subsequent translocation across the membrane is substantially increased (64-fold for d-galactose and 163.3-fold for d-glucose). These data indicate that Asn394 is associated with both the cytochalasin B and internal sugar binding sites. This conclusion is also supported by data showing that the sugar specificity of the mutant protein has been altered for d-xylose. This work powerfully illustrates how comparisons of the aligned amino acid sequences of homologous membrane proteins of unknown structure and characterization of their phenotypes can be used to map substrate and ligand binding sites.

The identification of transporter amino acid residues involved in the interaction with antibiotics will help elucidate the mechanism of recognition of both antibiotics and substrates. To this end, we have exploited the homology of the bacterial and mammalian transporters and compared their aligned amino acid sequences with their reported sensitivities to inhibition by cytochalasin B; it is probable that conserved residues are important in maintaining a common structure and mechanism and that differences are associated with variations in sugar specificity and antibiotic binding. In previous studies the covalent photolabeling of GLUT1 with cytochalasin B has been localized, by peptide mapping experiments, to a region containing amino acids Phe 389 to Trp 412 (30 -33, 39), which is proposed to form part of TM11 and the cytoplasmic loop joining it to TM10. Site-directed mutagenesis experiments on GLUT1 (40) led to a model where cytochalasin B can photolabel either Trp 388 or Trp 412 . These regions of the aligned sequences of GalP, AraE, XylE, GLUT1, GLUT2, GLUT3, GLUT4, GLUT7, and a high affinity D-fructose transporter, GLUT5 (41), were compared. The transporters GalP, AraE, GLUT1, GLUT2, GLUT3, and GLUT4 have a conserved Asn residue and are all susceptible to inhibition by cytochalasin B. In contrast, the transporters XylE, GLUT5, and GLUT7 do not have a conserved Asn residue and show no (XylE 2 and GLUT5 (41)) or little (GLUT7 (42)) sensitivity to inhibition by cytochalasin B. We therefore predicted that the conserved Asn residue would be involved in cytochalasin B binding. To test this hypothesis Asn 394 of GalP, which is predicted in a three-dimensional model to be in the middle of TM11 (Fig. 1), was changed by site-directed mutagenesis to Gln. The ability to over-express wild-type and mutant GalP proteins in high yield in appropriate E. coli strains has enabled us to achieve a rigorous characterization, which shows that changing Asn 394 to Gln markedly diminishes sensitivity to the antibiotic cytochalasin B, but not to forskolin, and also diminishes binding of sugar to the inward-facing side of the protein.

MATERIALS AND METHODS
Bacterial Strains-E. coli strain JM1100 HfrC his-gnd ⌬ thyA galK ptsM galP mglP ptsF ptsG (43), containing plasmids with wild-type or mutated galP genes, was the host strain for over-production of the GalP protein. After the mutagenic reaction (see below) the recombinant bacteriophage M13 DNA was transfected into E. coli strain TG1 ⌬ (lac-proAB) supE thi hsd ⌬ 5 FЈ[traD36 proAB ϩ lacI q lacZ ⌬M15]. The modification (Ϫ) and restriction (Ϫ) phenotype of strain TG1 necessitated that the mutant plasmids constructed from DNA isolated from strain 2  The Asn residue is predicted to be in the middle of TM11. The structures of the sugar substrate D-galactose and of the inhibitory antibiotics cytochalasin B and forskolin are also shown. TG1 were transformed into strain DH1 (modification (ϩ), restriction (Ϫ)) before introduction into E. coli strain JM1100 (modification (ϩ), restriction (ϩ) phenotype), to prevent restriction of unmodified plasmid DNA. E. coli strain DH1 has the genotype supE44 hsdR17 recA1 endA1 gyrA96 thi-1 relA1.
Growth of the Over-producing E. coli Strains-The E. coli strains JM1100(pPER3) (wild-type GalP (2)) and JM1100(pTPM6) (GalP with the Asn 394 3 Gln mutation, this work) were used for the constitutive over-expression of the wild-type and mutated GalP proteins. To gain maximum expression of GalP, strains were grown overnight in minimal media with 15 g/ml tetracycline, supplemented with L-histidine (80 g/ml) and thymine (20 g/ml). For sugar transport and sugar-H ϩ symport experiments, requiring lower levels of expression, cells were grown on rich media (2TY supplemented with 20 mM glycerol, 20 g/ml thymine, and 15 g/ml tetracycline).
Preparation of Inner Membranes Containing Over-expressed Protein-The E. coli cells were disrupted by explosive decomposition in a French Press at 137.5 MPa, and the inner membranes were prepared essentially as described by Osborn et al. (44). This procedure yields predominantly inside-out vesicles (45).
Quantification of Over-expressed Protein-A sample of the membrane preparation (30 g of protein) was subjected to SDS-polyacrylamide gel electrophoresis. After staining with Coomassie Blue, gels were scanned with a Molecular Dynamics 100A Computing Densitometer, and the proportion of GalP protein was measured. The levels of expression of the wild-type and mutant proteins were determined by comparison of the intensities of the bands corresponding to the GalP proteins on Coomassie-stained SDS-polyacrylamide gels. The expression of the Asn 394 3 Gln mutant protein was 107% that of the wild-type protein.
Protein Assays-The concentration of protein in the membrane preparations was assayed by the method of Schaffner and Weissman (46).
Oligonucleotide-directed, Site-specific Mutagenesis-The codon (AAC) for asparagine 394 of the galP gene was replaced by that of glutamine (CAG) as follows. The single-stranded DNA template for mutagenesis was prepared by subcloning a 2.46-kilobase pair AlwI-AlwI DNA fragment coding for the C-terminal half of GalP from plasmid pPER3 (which confers elevated expression of GalP (2)) into bacteriophage M13mp18. Mutagenesis was carried out with the Amersham Corp. in vitro mutagenesis kit using the mutagenic primer 5Ј-GGCAATCCACTGGGTGGCAGTG-3Ј (the mismatches are underlined) complementary to the coding strand of galP. The base changes were confirmed by single-stranded dideoxynucleotide sequencing. A doublestranded 1.561-kilobase pair MluI-NdeI fragment containing the base changes was isolated from the bacteriophage M13 construct and substituted for the wild-type DNA in MluI-NdeI cut plasmid pPER3 to give plasmid pTPM6 (Asn 394 3 Gln). The substitution was confirmed by subcloning the DNA containing the mutation back into bacteriophage M13mp18 and dideoxynucleotide sequencing. Double-stranded sequencing of the entire mutant galP gene in the expression plasmid confirmed that there were no other base changes in the galP gene sequence.
Sugar Transport Measurements-The transport of radioisotope-labeled sugars into intact cells was measured after the cells were energized with 10 mM glycerol as described by Henderson et al. (43). All transport measurements were carried out on cells that had been grown on rich media producing levels of GalP expression less than 1% total membrane protein. The level of expression of the mutant GalP protein was very similar to that of wild type judging by Western blots (data not shown). The velocity of D-galactose uptake (with D-galactose at 50 M) without the addition of inhibiting sugar did not vary greatly between experiments showing little day to day variation (wild type 24.3 Ϯ 2.8 nmol/mg/min (n ϭ 10) and Asn 394 3 Gln 6.73 Ϯ 0.88 nmol/mg/min (n ϭ 10)).
Sugar-H ϩ Symport Measurements-D-Galactose-promoted pH changes (sugar-H ϩ symport) were measured using 3.3 mM sugar with energydepleted anaerobic suspensions of intact cells in 150 mM KCl, 2 mM glycylglycine, pH 6.6, as detailed by Henderson and Macpherson (47). Initial velocities of D-galactose uptake into E. coli strains expressing wild-type and mutant GalP proteins were measured after the cells had been aerated in the presence of glycerol (10 mM) and a range of sugar concentrations for 3 min.
Permeabilization of the Cell Outer Membrane for Inhibition Studies-Cells were made permeable to cytochalasin B and forskolin by treatment with Tris-EDTA (48). Cells were grown for uptake experiments as Fluorescence Studies-Fluorescence spectra were measured in a Perkin-Elmer LS50B spectrophotofluorimeter. The protein was excited at either 280 or 297 nm and the fluorescence emission monitored between 300 and 400 nm. Rapid reactions were followed using an Applied Photophysics (London, UK) spectrophotofluorimeter, operated at 20°C, as described by Walmsley et al. (14,17,50). Fluorescence measurements were carried out in 50 mM potassium phosphate, 100 mM NaCl, and 1 mM EDTA, buffered to pH 7.4 at 20°C. Routinely the membranes were used at a protein concentration of 200 g/ml, in the above buffer.
Equilibrium Dialysis-Equilibrium dialysis was carried out as described by Walmsley et al. (50). The binding of 12-[ 3 H]forskolin and 4-[ 3 H]cytochalasin B to inner membranes (1.0 mg/ml) containing GalP was measured over the range of 0.05-80 M ligand at 4°C. Ratios of bound to free ligand were calculated from the equilibrium distribution of the radiolabeled ligand and used to determine both the K d and the number of ligand binding sites by an unweighted nonlinear leastsquares fit of the data to an hyperbola using the Biosoft program Ultrafit 2.1.
Statistical Analyses-The apparent K m values, with their standard deviations, for transport of radiolabeled sugar were obtained by a leastsquares fit of the unweighted initial rate data directly to a hyperbola FIG. 2. Energized transport catalyzed by the Asn 394 3 Gln mutant protein is mildly impaired. E. coli strain JM1100 expressing the wild-type and mutant proteins were grown in 2 TY supplemented with 20 mM glycerol until A 680 ϭ 0.6. The cells were harvested and washed twice in the growth volume of 150 mM KCl, 5 mM MES, pH 6.6, and finally resuspended to a density of 0.68 mg of dry cell mass/ml. Aerated samples (0.25 ml) were incubated at 25°C for 3 min with 25 mM glycerol before addition of 6.25 l of radiolabeled sugar to give the indicated concentration range. After 15 s, 2 ml was filtered rapidly and washed four times in 2 ml of the above medium. The initial rates of sugar uptake (nmol/min per dry mass) were calculated and displayed as a Lineweaver-Burk plot. The graph is a typical example of the uptake of 1-[ 3 H]D-galactose catalyzed by wild-type and mutant proteins. The data yielded K m values of 58.9 Ϯ 8.6 and 107.6 Ϯ 34.0 M and V max values of 65.4 Ϯ 2.3 and 28.5 Ϯ 2.2 nmol/mg/min for wild-type and mutant proteins, respectively. Similar experiments were carried out on the uptake of D-glucose and D-xylose, and the results are shown in Table I. using Biosoft program Ultrafit 2.1. Generally, rates of the quench in the intrinsic fluorescence of GalP, when it was mixed with the antibiotics in a stopped-flow apparatus, were fitted to an exponential function using the non-linear least-squares regression program supplied with the Applied Photophysics SF.17MV stopped-flow equipment. The data sets, for the concentration dependence of the rate of binding of the antibiotics, were analyzed by fitting to the appropriate equation using the non-linear least-squares regression program SIGMA PLOT (Jandel Scientific).

Substitution of Asn 394 by Gln Does Not Greatly Impair the
Sugar Transport Activity of GalP-The transport of D-galactose by the wild-type GalP protein, under energized conditions, was characterized by a hyperbolic increase in the initial rate with increasing concentrations of sugar. A least-squares fit yielded apparent K m and V max values of 42.4 -58.9 M and 59.1-65.4 nmol/mg/min ( Fig. 2 and Table I). The K m and V max values quoted here and subsequently are apparent as it was not known whether the co-substrate, H ϩ , was at saturating amounts. The substitution of Asn 394 by Gln led to a 2-fold increase in the K m for D-galactose and a 2-fold decrease in the V max , compared with the wild type ( Fig. 2 and Table I). The data indicated a relatively small (4.8-fold) reduction in the specificity (as defined by V max /K m ) of the mutant protein for D-galactose relative to the wild type. The uptake of 50 M D-galactose was accumulated by the mutant to 42-fold that of the external media, compared with 88-fold by the wild type. These data demonstrated that the mutant protein can catalyze energized transport and that the gross conformation of the protein is not significantly impaired. D-Glucose was transported into the mutant with a V max 1.5-fold higher than that of the wild-type protein, although the K m was 3.3-fold higher ( Table I). The transport of D-xylose was characterized by a 4.3-fold increase in the K m and a 0.8-fold decrease in the V max compared with that of the wild type. The V max /K m values were 2.1-fold (D-glucose) and 5.3-fold lower (D-xylose) than that of the wild type.
The Asn 394 3 Gln Mutant Protein Still Catalyzes D-Galactose-Proton Symport-The sugar-H ϩ proton symport activities of the wild-type and mutant proteins were assayed by monitoring D-galactose-promoted alkaline pH changes (data not shown), which are diagnostic of sugar-H ϩ symport (51,52). The extent of D-galactose-H ϩ symport catalyzed by the mutant protein (5.19 Ϯ 2.7 nmol of H ϩ per mg (n ϭ 5)) was not significantly different from that of the wild-type protein (4.87 Ϯ 3.88 nmol of H ϩ per mg (n ϭ 8)). However, the rate of D-galactose H ϩ -symport catalyzed by the mutant (1.16 Ϯ 0.41 nmol of H ϩ per mg (n ϭ 5)) was 2.8-fold lower that of the wild type (3.31 Ϯ 1.54 nmol of H ϩ per mg (n ϭ 8)). Again this implies that the gross conformation and transport competence of the mutant protein are not seriously affected by the Asn 394 3 Gln mutation.
D-Galactose Transport Catalyzed by GalP with the Asn 394 3

Gln Mutation Was Inhibited by Forskolin but Not by Cytochalasin B-
The abilities of cytochalasin B or forskolin to inhibit the energized uptake of 50 M D-galactose catalyzed by wildtype and mutant GalP into E. coli cells were investigated. The cell walls were rendered permeable to the antibiotics by prior treatment with Tris-EDTA, pH 8.0 (see "Materials and Methods"). In the case of the wild type, both cytochalasin B (80 M) and forskolin (80 M) inhibited the transporter, cytochalasin B being a slightly more potent inhibitor (Fig. 3). However, the transport catalyzed by the mutant protein was not significantly inhibited by cytochalasin B, although it demonstrated a marked increase in sensitivity to inhibition by forskolin.

The Asn 394 3 Gln Mutant Protein Was Photolabeled by IAPS-Forskolin but Not by Cytochalasin B-
The wild-type GalP protein was susceptible to photolabeling with 4-[ 3 H]cytochalasin B and 3-125 I-APS-forskolin, and the protein was protected against this photolabeling by the physiological substrate, D-galactose, but not by L-galactose, which is not a substrate (Fig. 4, lanes 2 and 3). This protection by substrate showed that both cytochalasin B and IAPS-forskolin specifically label the GalP protein (13-15).

TABLE I
The kinetic parameters K m and V max for the transport of D-galactose, D-glucose, and D-xylose catalyzed by the wild-type and Asn 394 3 Gln mutant GalP proteins Initial rates of the transport of the indicated radiolabeled sugar were measured over an appropriate range of concentrations, from about 0.5 K m to 5 K m . The maximal rate of transport (V max ) and the K m were determined by a least-squares fit of each data set to a Michaelis-Menten equation.

Parameter
Wild-type Asn 394 3 Gln 0.0768 nmol/mg/min/M 0.0146 nmol/mg/min/M The Asn 394 3 Gln mutant GalP protein was photolabeled by 3-125 I-APS-forskolin, and D-galactose, but not L-galactose, protected the protein against the ligand; the extent of labeling and of protection were at levels similar to those of the wild-type protein (Fig. 4, B and D). However, no photolabeling of the mutant protein by cytochalasin B was detected (Fig. 4, A and C).
The Asn 394 3 Gln Mutant Protein Binds Forskolin but Not Cytochalasin B-The equilibrium binding of the antibiotics cytochalasin B and forskolin to GalP over-expressed in E. coli is characterized by linear Scatchard plots indicating a single species of high affinity binding sites; furthermore, one unlabeled antibiotic displaces the other labeled one in a simple competitive manner (2,15). When the data were fitted to a single hyperbolic function, by least-squares analysis (53), they revealed dissociation constants (K d ) for cytochalasin B binding of 4.5 Ϯ 1.1 M (n ϭ 10) and for forskolin binding of 1.36 -1.43 M (2, 15). The binding sites were measured at 12.8 Ϯ 2.9 nmol/mg membrane protein (n ϭ 10) and 7.2-8.1 nmol/mg membrane protein for cytochalasin B and forskolin, respectively (2,15). These values were compared with the amount of expressed GalP as determined by quantitative densitometry of SDS-polyacrylamide gel electrophoresis gels (2, 54) and indicated a 1:1 ratio for the binding of the antibiotics to the GalP protein.
To determine the concentration of the binding sites and the The binding of the antibiotics cytochalasin B and forskolin to the over-expressed wild-type GalP protein was characterized by a quench in the intrinsic protein fluorescence. This has previously allowed us to monitor the binding of these antibiotics by a combination of steady-state and stopped-flow spectrofluorimetric techniques, providing a rigorous kinetic analysis of the binding process (14, 16). To assess the importance of Asn 394 in the binding of antibiotic, the intrinsic fluorescence of the mutant protein was titrated with antibiotic over a range of concentrations up to 80 M (Figs. 6 and 7). A fit of the data to a hyperbola yielded an overall dissociation constant (K d ) for forskolin binding of 1.48 Ϯ 0.5 M (n ϭ 3), similar to that of 1.83 Ϯ 0.56 M determined for the wild type (16). This is consistent with the equilibrium dialysis data (above), which showed similar K d values for the binding of forskolin to both wild-type and mutant proteins. In contrast to the equilibrium dialysis and photolabeling experiments described above, some low affinity cytochalasin B binding to the mutant protein was detected using the fluorescence method; however, this was shown to be with an affinity 37-and 53-fold lower than the wild-type protein (K d 41.4 and 58.5 M) in two separate experiments.
The extent of the quenching of fluorescence by saturating amounts of forskolin (10 M) was compared between the wildtype and Asn 394 3 Gln mutant proteins. Measurements were made with ex at 280 and 297 ( em max 330 -340 nm) to detect any differences due to light absorption by tyrosine and/or tryptophan residues and at two protein concentrations (50 and 100 g/ml) to detect any differences due to the light scattering properties of the membrane preparations; no differences in the level of quench was found at either wavelength or between the two protein concentrations in either preparation. A 1.4-fold increase in the fluorescence quench of the mutant protein, relative to the wild type (wild type 8.09 Ϯ 0.32% (n ϭ 7); Asn 394 3 Gln 11.36 Ϯ 0.88% (n ϭ 6)), was observed with ex 280 and 1.8-fold increase with ex 297 (wild type 6.16 Ϯ 0.49% (n ϭ 5); Asn 394 3 Gln 11.06 Ϯ 0.14% (n ϭ 4)). A similar quantification of the amount of fluorescence quench concomitant with the binding of cytochalasin B to the mutant protein could not be made as saturating amounts of ligand could not be obtained.
Measurement of the Transient Kinetic Parameters for the Binding of Antibiotics to the Asn 394 3 Gln Mutant Protein- The binding of either forskolin or cytochalasin B to the GalP protein was previously analyzed by stopped-flow fluorescence spectroscopy in terms of the following three-step Model A (14,16,38).
T o and T i are, respectively, the outward-and inward-facing forms of the unloaded transporter, and T i -antibiotic is the transporter-antibiotic complex. A further intermediate conformational change of the transporter-antibiotic complex was also proposed (T i/o -antibiotic). T i/o is possibly a state in which the transporter is attempting to undergo reorientation and thus is a high affinity T-antibiotic complex.
For such a model, the dissociation constant of the transporter-forskolin complex is given by Equation 1.
where K 1 ϭ k Ϫ1 /k 1 , K 2 ϭ k Ϫ2 /k 2 , and K 3 ϭ k Ϫ3 /k 3 . The Asn 394 3 Gln GalP mutant protein was characterized by a concentration-dependent linear increase in the rate of the fast phase of forskolin binding to the T i conformation of the protein (Fig. 6), indicating association (k 2 ) and dissociation rate constants (k Ϫ2 ) of 1.51 Ϯ 0.1 M Ϫ1 s Ϫ1 and 2.37 Ϯ 0.96 s Ϫ1 , respectively; the dissociation constant (K 2 (k Ϫ2 /k 2 )) was calculated to be 1.57 Ϯ 0.64 (Table II). The rates of association and dissociation of forskolin with the mutant were 4-and 3.6-fold slower, respectively, than for the wild type. Thus, the dissociation constant (K 2 ) of the mutant is almost identical to the wild type, demonstrating that the affinity of the T i or putative inward-facing transporter of the mutant protein for forskolin is unchanged. As K 2 and overall K d values are similar for both wild-type and mutant proteins there is no evidence to suggest changes to either K 1 or K 3 .
Cytochalasin B also bound to the mutant protein in a linear, concentration-dependent manner (Fig. 7), indicating an association rate constant of 0.67 Ϯ 0.28 M Ϫ1 s Ϫ1 . In previous work with the wild-type GalP protein the dissociation rate constant for cytochalasin B was measured by directly displacing ligand, bound to the transporter, by adding sugar substrate (49). The transport protein was previously equilibrated with 80 M ligand and mixed with 500 mM sugar in the stopped-flow apparatus to displace the bound ligand. The increase in protein fluorescence resulting from displacement of the bound cytochalasin B by D-galactose could be fitted to a single exponential function revealing a dissociation rate constant of 12.18 Ϯ 2.78 s Ϫ1 . The dissociation constant K 2 (k Ϫ2 /k 2 ) for the T i conformation of the mutant protein was calculated as 18.1 Ϯ 8.78 M. This value is 44-fold higher than that of the wild type and is similar to the 37-53-fold decrease in the overall affinity (increase in overall K d ) for cytochalasin B.
The Binding of D-Galactose and D-Glucose to the Internal Binding Site Is Greatly Impaired in the Asn 394 3 Gln Mutant of GalP-8-Anilino-1-naphthalene sulfonate (ANS) can be used as a fluorescent probe to monitor conformational changes in the GalP protein induced by the binding of sugar (17). Transported sugars such as D-galactose and D-glucose cause an enhancement in the ANS fluorescence; non-transported sugar analogues and antibiotics produce only a slight quench in the ANS fluorescence, but they can reverse the enhancement in fluorescence induced by transported sugars. The increase in ANS fluorescence is attributable to the sugar-induced reorientation of the transporter from an inward to an outward-facing conformation. Non-transported ligands are thought to reverse the fluorescence enhancement by recruiting the transporter to an inward-facing conformation. Before interpreting the effects of the Asn 394 3 Gln mutation, we first considered the kinetic model for the wild-type protein, which was derived from measurements of ANS fluorescence (17).
The rate of enhancement in ANS fluorescence produced by D-galactose was measured by stopped-flow spectrofluorimetry (17) and was found to increase in a biphasic manner as [sugar] increased. The fast phase was attributed to the initial binding of the sugar to the inward-facing conformation of the protein and the slow phase to a subsequent conformational change. The rate of the fast phase increased hyperbolically with increasing D-galactose concentration indicative of the rapid bind-ing of the transporter that was rate-limited by a slower conformational change at near saturating concentrations (see Equation 2). sugar ϩ T i L | ; Where T i and T o are inward-and outward-facing conformational forms of the transporter, respectively; K D-gal is the dissociation constant for the inward-facing GalP-D-galactose complex and K 2 ϭ k 2 /k Ϫ2 ; k 2 and k Ϫ2 are rate constants for the conformational change subsequent to sugar binding and were attributed to transporter reorientation. The change in fast phase was fitted to the following hyperbolic function as shown in Equation 3. .
However, it was also measured from the total fluorescence change since the sum of the fast and slow fluorescence phases also exhibited hyperbolic dependence upon the D-galactose concentration with an overall K d of 0.66 Ϯ 0.033 mM. As the calculated value for the overall K d is greater than the measured value, a further conformational change following the formation of T o -D-gal causing additional tightening of the binding of Dgalactose was proposed. The calculated K d for the binding of glucose (0.33 mM) was also greater than the measured value (0.18 mM). These results led to the suggestion that the kinetic scheme shown above be expanded to give Equation 5.
Where T o * is an outward-facing conformation, slightly different to that of T o .
Hence the K d would be as shown in Equation 6.
In contrast to the wild-type protein, binding of D-galactose and D-glucose to the mutant protein was characterized by a monophasic increase in the ANS fluorescence with time. The rate was apparently independent of sugar concentration, and it was not possible to determine rate constants, k 2 and k Ϫ2 , and the dissociation constant, K D -gal. It is probable that k 2 and k Ϫ2 are very similar. The total fluorescence enhancement, which was 8.5 times lower in the case of D-galactose and 3.6 times lower in the case of D-glucose, did, however, exhibit a hyperbolic dependence upon the concentration of sugar (Fig. 8). The concentration dependence of the total fluorescence enhancement for D-galactose and D-glucose fitted to a hyperbolic function and from three separate experiments yielded overall K d values of 42.3 Ϯ 11.8 for D-galactose and 29.4 Ϯ 3.5 for Dglucose. These K d values are a 64.1-and 163.3-fold increase of the wild type for D-galactose and D-glucose, respectively. As it was possible only to measure the overall K d for sugar binding, which encompasses the initial binding event to the inwardfacing conformation (T i ) and the translocation of sugar across the membrane (see three-step kinetic Model A above), it cannot be ruled out that the mutation has affected any of the subsequent reorientation steps. However, the energized transport of D-glucose and D-galactose catalyzed by the mutant protein into whole cells is not severely affected, implying that the sugar translocation mechanism remains intact and that it is the binding of sugar to the internal binding site that is predominantly impaired.
Substrate Initial velocities of D-galactose uptake catalyzed by the wildtype and mutant proteins into E. coli were measured. The 1/v against [I] plots (Dixon plots) for D-galactose were linear, as expected for simple hyperbolic binding, in the presence of unlabeled D-galactose, 2-deoxy-D-glucose, D-talose, 6-deoxy-D-glucose, 2-deoxy-D-galactose and D-mannose. At higher concentrations of D-xylose, 6-deoxy-D-galactose (wild type only), D-glucose, and L-arabinose the Dixon plots were non-linear and deviated systematically from hyperbolic binding (data not shown). However, the data points at the lower concentrations of these inhibiting substrates did display linearity, and extrapolated estimates of K i were determined. It was reported previously (13) that the kinetics of transport of D-glucose, 2-deoxy-D-glucose, and 6-deoxy-D-galactose transport by GalP were nonhyperbolic. In order that changes in substrate specificity of the mutant protein could be readily detected, a ratio of mutant K i to wild-type K i was calculated, which was then normalized by dividing with the ratio for D-galactose (Table III). The recognition by the mutant protein of most of the sugar substrates was only altered from that of the wild type between 0.5-2.0-fold, indicating little change in sugar selectivity. Surprisingly, the specificity for D-xylose was seven times lower with the mutant protein compared with the wild type. There is no obvious, simplistic interpretation of this effect such as an altered substrate recognition in terms of a single H-bonded sugar-protein interaction. Clearly the mutation of the Asn 394 residue to Gln has, in terms of D-xylose, selectively affected the substrate specificity of the GalP protein.

DISCUSSION AND CONCLUSIONS
An alignment of the amino acid sequences of those proteins that are homologous to GalP, together with a knowledge of the ability of these transporters to bind cytochalasin B, suggested that the Asn 394 residue might be involved in the recognition of cytochalasin B as a part of the cytochalasin B binding site or pocket. For the purposes of this work we will define the binding site as comprising those amino acids that bind directly with the antibiotic, and the binding pocket is defined as amino acids in the immediate environment of the antibiotic that do not necessarily bind directly to it. To investigate the involvement of Asn 394 , the residue was altered by mutagenesis to Gln.
Inward D-galactose transport catalyzed by the mutant protein, as defined by the parameters K m and V max , was relatively unimpaired, implying that the gross conformation of the pro-tein was intact and the transport mechanism conserved. Similarly, the mutant protein was able to catalyze sugar proton symport, to an extent similar to wild type albeit at a 2.8-fold lower rate, indicating that coupling between proton and sugar transport was maintained.
Previous equilibrium binding experiments on the wild-type protein revealed mutual competition between cytochalasin B and forskolin for the antibiotic binding site (16). It was therefore of interest to investigate the effects of the Asn 394 3 Gln substitution on the interaction of GalP with both antibiotics. The mutant protein was photolabeled by IAPS-forskolin at levels similar to that of the wild-type protein, and the reaction was protected by D-galactose. These data showed that Asn 394 is not essential for photolabeling by forskolin. However, it cannot be ruled out that Asn 394 of wild-type GalP is the photolabeled amino acid residue and that Gln 394 is being photolabeled in the mutant protein. Positive identification must wait for the direct sequencing of protein fragments generated from photolabeled transporter. A Scatchard analysis demonstrated that the overall affinity for forskolin binding to the mutant protein and the number of forskolin binding sites were almost identical to those of the wild-type protein; this unchanged overall affinity of the mutant protein for forskolin was confirmed by the titration of the protein's fluorescence. D-Galactose transport catalyzed by the mutant protein demonstrated a marked increase in sensitivity to inhibition by forskolin compared with the wild type (wild type 58.0 Ϯ 2.9% and Asn 394 3 Gln 7.3 Ϯ 2.9% compared with the ethanol control). This increase probably reflects a decrease in the level of competition by D-galactose. The maximal fluorescence quench induced by forskolin binding was 1.8fold greater with the mutant protein than the wild type ( ex 297; em max 330 -340 nm), although the amount of GalP was the same. This indicates that the substitution of Asn 394 by Gln altered the environment of one or more tryptophan residues. Characterization of a Trp 395 3 Phe mutant GalP protein indicated that Trp 395 is in a conformationally active region of the protein and that this residue is the main reporter of forskolin binding in fluorescence quenching experiments (38). The increase in the maximal fluorescence quench due to the binding of forskolin to the Asn 394 3 Gln mutant can be rationalized by suggesting that the presence of Gln 394 causes a localized conformational change altering the environment of the neighboring Trp 395 residue. Although the overall affinity for forskolin binding to the mutant protein is unaffected, transient fluorescence measurements showed that the rates of association (k 2 ) and dissociation (k Ϫ2 ) to the inward-facing conformation of the protein (T i ) are reduced (3.9-and 3.6-fold for k 2 and k Ϫ2 , re- spectively). The affinity for the initial interaction with forskolin to the inward-facing conformation of the protein (T i ), as defined by the dissociation constant K 2 (k Ϫ2 /k 2 ), was unchanged. As K 2 and overall K d values are similar for both wild-type and mutant proteins, there is no evidence to suggest changes to either K 1 or K 3 .
In stark contrast to the binding of forskolin, cytochalasin B was shown, in fluorescence quenching experiments, to bind to the mutant protein with an overall affinity 37-53-fold lower than that of the wild-type protein. The displacement of cytochalasin B from the mutant protein by sugar substrate provided corroborating evidence of ligand binding. This low affinity cytochalasin B binding was not detectable at all in photolabeling or equilibrium dialysis experiments, most likely due to an insufficient concentration or specific activity of the radiolabeled cytochalasin B.
Transient fluorescence measurements showed that the affinity for the initial interaction with cytochalasin B to the inwardfacing conformation of the protein (K 2 , as defined by k Ϫ2 /k 2 ) was also much lower than wild type (40-fold lower). The reduction in overall affinity for cytochalasin B cannot be attributed to stabilization of the T o (outward-facing) conformation of the mutant transporter, otherwise a similar effect should be observed with forskolin binding; thus, it is most likely to be entirely due to a reduction in affinity of the T i conformation. Cytochalasin B was shown to have a rate of association to the mutant protein (k 2 ) lower than that of the wild type (9-fold) and as such is similar to forskolin. However, in direct contrast with the rate of forskolin dissociation, that of cytochalasin B from the mutant protein is higher (4.9-fold) than that of the wild type.
Possible explanations for these data are that the amino acid substitution has 1) altered the shape of the forskolin binding pocket making it more difficult for the antibiotic to bind, but once bound it is held within the pocket for longer, and (2) disrupted a single H-bonded cytochalasin B-protein interaction resulting in a reduction of overall affinity for the ligand. The reduction in affinity for cytochalasin B and alteration of forskolin binding provides evidence that the cytochalasin B and forskolin binding sites are closely associated. We speculate that Asn 394 forms part of the cytochalasin B binding site and part of the forskolin binding pocket. However, the possibility that the disruption in cytochalasin B binding is due to steric hindrance by the larger Gln residue cannot be discounted.
In contrast to the energized transport of sugar, which is relatively unimpaired, ANS fluorescence measurements show that the overall K d describing the binding of D-galactose and D-glucose to the inward-facing conformation of the mutant protein and their subsequent translocation across the membrane were drastically higher than that for the wild-type protein (64.1-fold for D-galactose and 163.3-fold for D-glucose). The fact that energized transport catalyzed by the mutant transport is relatively unimpaired suggests that the translocation mechanism is intact. It is therefore most likely that the decrease in overall affinity is largely due to loss of affinity for these sugars at the inward-facing binding site.
The substitution of the Asn 394 residue by Gln has, at least, at the primary structural level made the GalP protein more like XylE. Such single residue differences may be the means by which proteins are able to discriminate between different substrates, e.g. between hexoses and pentoses. The sugar specificity of the wild-type GalP protein has been extensively investigated (2,13,15,17), which showed the importance of the C-3 hydroxyl and C-6 on the pyranose ring for hexose recognition by GalP and the lack of importance of the C-2 and C-4 hydroxyl groups. These observations parallel the requirements for sugar recognition by the passive glucose transporters of human erythrocytes (GLUT1) and rat adipocytes (GLUT4) (11,12), confirming the functional similarity of the bacterial and mammalian transporters. The substrate specificity study carried out for energized uptake of sugars in this work did not reveal a general role for the Asn 394 residue in the recognition of external sugar substrates, but, surprisingly, the specificity for Dxylose was significantly reduced (7.6-fold compared with the wild type); there is no obvious, simplistic interpretation of this effect, such as an altered substrate recognition in terms of a single H-bonded sugar-protein interaction. Overall, these observations support the other data that indicate Asn 394 is part of the internal binding site for sugars.
Cytochalasin B has been shown by kinetic analysis to compete with D-glucose for binding to the inward-facing conformation of the GLUT1 protein (55). Our data, which demonstrate that the disruption of sugar binding to the inward-facing conformation of GalP (as opposed to the outward-facing conformation) is coupled to the disruption of cytochalasin B binding by the Asn 394 3 Gln mutation, provide further evidence of the functional and structural similarity of the GLUT1 and GalP proteins. These data contribute to the ongoing debate as to whether cytochalasin B and sugar bind to the same site of the inhibitor susceptible proteins and suggest, at least with GalP, that this is the case.
An Asn 415 3 Asp mutant of the GLUT1 transporter has been isolated (56). Asn 415 is conserved in all the GalP, AraE, XylE, and GLUT transporters and is the 4th residue after Asn 394 in GalP. Characterization of the Asn 415 3 Asp mutant GLUT1 protein showed 40% less cytochalasin B photolabeling although the inhibition of labeling by ethylene glucose, which preferentially binds to the outer glucose binding site (57), inhibited cytochalasin B similar to the wild type. This led to the suggestion that the Asn 415 residue is likely to reside close to the inner glucose and cytochalasin B binding sites of GLUT1. It is interesting to note that to make a complete turn of an ␣-helix, the polypeptide backbone must traverse 3.6 amino acids. Thus amino acids three or four amino acids apart (as in the case of Asn 412 (aligned with Asn 394 of GalP) and Asn 415 of GLUT1) would be expected to project from the same side of an ␣-helix. It has also been shown (38) that the affinity for the initial interaction of cytochalasin B to a Trp 395 3 Phe mutant as described by the apparent dissociation constant (K 2 ) is 43-fold lower than that for the wild type while the K 2 for forskolin binding to the mutant is similar to wild type. These data suggest that the residue adjacent to Asn 394 (Trp 395 ) is also associated with the cytochalasin B but not the forskolin binding site. Interestingly, the affinity for D-galactose of the inwardfacing binding site of the Trp 395 3 Phe mutant is similar to wild type and is therefore unlikely to be involved in D-galactose binding (38). It is by the collation of such data from many mutants that structural and mechanistic models for these related sugar transporters will begin to emerge, and the binding sites of sugar and antibiotic will be determined.