Asparagine 394 in Putative Helix 11 of the Galactose-H1 SymportProtein (GalP) from Escherichia coli Is Associated with the InternalBinding Site for Cytochalasin B and Sugar*

(Received for publication, August 20, 1996, and in revised form, February 10, 1997)

Terence P. McDonald‡, Adrian R. Walmsley§¶, and Peter J. F. Henderson‡i

From the ‡Department of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT and the §KrebsInstitute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield,P.O. Box 594, Firth Court, Western Bank, Sheffield, S10 2UH, United Kingdom

The galactose-H1 symport protein (GalP) of Esche-richia coli is very similar to the human glucose trans-port protein, GLUT1, and both contain a highly con-served Asn residue in predicted helix 11 that is differentin a cytochalasin B-resistant member of this sugar trans-port family (XylE). The role of the Asn394 residue (whichis predicted to be in putative trans-membrane a-helix11) in the structure/activity relationship of the D-galac-tose-H1 symporter (GalP) was therefore assessed bymeasuring the interaction of sugar substrates and theinhibitory antibiotics, cytochalasin B, and forskolinwith the wild-type and Asn394 3 Gln mutant proteins.Steady-state fluorescence quenching experiments showthat the mutant protein binds cytochalasin B with a Kd37–53-fold higher than the wild type. This low affinitybinding was not detected with equilibrium binding orphotolabeling experiments. In contrast, the mutant pro-tein binds forskolin with a Kd similar to that of the wildtype and is photolabeled by 3-125I-4-azido-phenethyl-amido-7-O-succinyl-desacetyl-forskolin. The mutantprotein displays an increased amount of steady-statefluorescence quenching with the binding of forskolin,suggesting that the substitution of the Asn residue hasaltered the environment of a tryptophan, probablyTrp395, in a conformationally active region of the pro-tein. Time-resolved fluorescence measurements on themutant protein provided association and dissociationrate constants (k2 and k22), describing the initial inter-action of cytochalasin B to the inward-facing bindingsite (Ti), that are decreased (9-fold) and increased (4.9-fold) compared with the wild type. This yielded a disso-ciation constant (K2) for cytochalasin B to the inward-facing binding site 44-fold higher than that of the wildtype. The binding of forskolin gave values for k2 and k223.9- and 3.6-fold lower, respectively, yielding a K2 valuefor Ti similar to that of the wild type. The low overallaffinity (high Kd) of the mutant protein for cytochalasinB is due mainly to a disruption in binding to the Ticonformation. It is proposed that Asn394 forms either adirect binding interaction with cytochalasin B or is part

of the immediate environment of the binding site andthat Asn394 is in the immediate environment, but notpart, of the forskolin binding site. The ability of themutant protein to catalyze energized transport is onlymildly impaired with 4.8- and 2.1-fold reduction inVmax/Km values for D-galactose and D-glucose, respec-tively. In stark contrast, the overall Kd describing bind-ing of D-galactose and D-glucose to the inward-facingconformation of the mutant and their subsequent trans-location 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 boththe cytochalasin B and internal sugar binding sites. Thisconclusion is also supported by data showing that thesugar specificity of the mutant protein has been alteredfor D-xylose. This work powerfully illustrates how com-parisons of the aligned amino acid sequences of homol-ogous membrane proteins of unknown structure andcharacterization of their phenotypes can be used to mapsubstrate and ligand binding sites.

The D-galactose-H1 symporter (GalP)1 from Escherichia coliis homologous to the L-arabinose-H1 symporter (AraE) and theD-xylose-H1 symporters (XylE) of E. coli, with 64 and 33%identity, respectively (1, 2). These transporters are also homol-ogous to a family of mammalian passive facilitated glucosetransporters (GLUT) (2–9). The inclusion of both passive andactive transporters in the homologous transporter family im-plies they share common features of both structure and molec-ular mechanism. Furthermore, the E. coli and mammalianproteins are predicted to have a similar membrane topology,comprising 12 membrane spanning a-helices (12 TM), withhelices 6 and 7 connected by a cytoplasmic domain containing60–70 amino acids (Fig. 1) (2, 8, 10). The sugar specificities ofthe E. coli transporters differ, with GalP primarily transport-ing hexoses and AraE and XylE pentoses. However, the sugarspecificity of GalP is very similar to that of the glucose trans-porters from human erythrocytes (GLUT1) and rat adipocytes(GLUT4) (11–14) giving rise to the suggestion that GalP is thebacterial equivalent of the mammalian glucose transporter (9).

This suggestion is reinforced by the observation that GalP-* This work was supported by research grants from the WellcomeTrust and Medical Research Council (MRC) (to P. J. F. H. and A. R. W.)and from the Science and Engineering Research Council (to P. J. F. H.)and by equipment grants from the University of Leeds (to P. J. F. H.),University of Sheffield (to A. R. W.) and the Royal Society (to A. R. W.).The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

¶ MRC Senior Fellow.i To whom correspondence should be addressed: Dept. of Biochemis-

try and Molecular Biology, University of Leeds, Leeds, LS2 9JT, UnitedKingdom. Tel.: 44 113 2333122; Fax: 44 113 2333167.

1 The abbreviations used are: GalP, the D-galactose-H1 symporter ofE. coli; AraE, the L-arabinose-H1 symporter of E. coli; XylE, the xy-lose-H1 symporter of E. coli; GLUT1, the human erythrocyte glucosetransporter; ANS, 8-anilino-1-naphthalene-sulfonate; TM, putativetrans-membrane a-helix; Km and Vmax are apparent values as it was notknown whether the co-substrate, H1, was at saturating amounts; MES,24-morpholineethanesulfonic acid; IAPS-forskolin, 3-125I-4-azido-phen-ethylamido-7-O-succinyl-desacetyl-forskolin; APS, azido-phenethyl-amido-7-O-succinyl.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 24, Issue of June 13, pp. 15189–15199, 1997© 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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mediated sugar transport is inhibited by the antibiotics cy-tochalasin B and forskolin (13–17), which are also potent in-hibitors of glucose transport mediated by GLUT1, GLUT2,GLUT3, and GLUT4 (18–29). The structures of these antibiot-ics are shown in Fig. 1. Both the GLUT1 and GalP proteins canbe photolabeled by cytochalasin B and a derivative of forskolin(IAPS-forskolin) in a substrate-protectable manner (2, 13, 15,16, 30–34). The binding of both cytochalasin B and forskolininduces a quench in the intrinsic protein fluorescence of bothGLUT1 and GalP (14, 16, 22, 35–37). This phenomenon hasallowed the kinetics of the binding of cytochalasin B to GLUT1and cytochalasin B and forskolin to GalP to be time-resolvedusing stopped-flow fluorescence spectroscopy; the binding ofcytochalasin B and forskolin to GalP and of cytochalasin B toGLUT1 occurs by similar mechanisms, with at least threeconformational states of the transport proteins identified(14, 16, 38).

The identification of transporter amino acid residues in-volved in the interaction with antibiotics will help elucidate themechanism of recognition of both antibiotics and substrates. Tothis end, we have exploited the homology of the bacterial andmammalian transporters and compared their aligned aminoacid sequences with their reported sensitivities to inhibition bycytochalasin B; it is probable that conserved residues are im-portant in maintaining a common structure and mechanismand that differences are associated with variations in sugarspecificity and antibiotic binding. In previous studies the cova-lent photolabeling of GLUT1 with cytochalasin B has beenlocalized, by peptide mapping experiments, to a region contain-ing amino acids Phe389 to Trp412 (30–33, 39), which is proposedto form part of TM11 and the cytoplasmic loop joining it toTM10. Site-directed mutagenesis experiments on GLUT1 (40)

led to a model where cytochalasin B can photolabel eitherTrp388 or Trp412. These regions of the aligned sequences ofGalP, AraE, XylE, GLUT1, GLUT2, GLUT3, GLUT4, GLUT7,and a high affinity D-fructose transporter, GLUT5 (41), werecompared. The transporters GalP, AraE, GLUT1, GLUT2,GLUT3, and GLUT4 have a conserved Asn residue and are allsusceptible to inhibition by cytochalasin B. In contrast, thetransporters XylE, GLUT5, and GLUT7 do not have a con-served Asn residue and show no (XylE2 and GLUT5 (41)) orlittle (GLUT7 (42)) sensitivity to inhibition by cytochalasin B.We therefore predicted that the conserved Asn residue wouldbe involved in cytochalasin B binding. To test this hypothesisAsn394 of GalP, which is predicted in a three-dimensionalmodel to be in the middle of TM11 (Fig. 1), was changed bysite-directed mutagenesis to Gln. The ability to over-expresswild-type and mutant GalP proteins in high yield in appropri-ate E. coli strains has enabled us to achieve a rigorous charac-terization, which shows that changing Asn394 to Gln markedlydiminishes sensitivity to the antibiotic cytochalasin B, but notto forskolin, and also diminishes binding of sugar to the in-ward-facing side of the protein.

MATERIALS AND METHODS

Bacterial Strains—E. coli strain JM1100 HfrC his-gndD thyA galKptsM galP mglP ptsF ptsG (43), containing plasmids with wild-type ormutated galP genes, was the host strain for over-production of the GalPprotein. After the mutagenic reaction (see below) the recombinant bac-teriophage M13 DNA was transfected into E. coli strain TG1 D(lac-proAB) supE thi hsdD5 F9[traD36 proAB1 lacIq lacZ DM15]. The mod-ification (2) and restriction (2) phenotype of strain TG1 necessitatedthat the mutant plasmids constructed from DNA isolated from strain

2 T. P. McDonald and P. J. F. Henderson, unpublished data.

FIG. 1. Two-dimensional model of the galactose-H1 symport protein. The Asn394 residue of GalP mutated to Gln in this study is indicated.The Asn residue is predicted to be in the middle of TM11. The structures of the sugar substrate D-galactose and of the inhibitory antibioticscytochalasin B and forskolin are also shown.

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TG1 were transformed into strain DH1 (modification (1), restriction(2)) before introduction into E. coli strain JM1100 (modification (1),restriction (1) phenotype), to prevent restriction of unmodified plasmidDNA. E. coli strain DH1 has the genotype supE44 hsdR17 recA1 endA1gyrA96 thi-1 relA1.

Growth of the Over-producing E. coli Strains—The E. coli strainsJM1100(pPER3) (wild-type GalP (2)) and JM1100(pTPM6) (GalP withthe Asn394 3 Gln mutation, this work) were used for the constitutiveover-expression of the wild-type and mutated GalP proteins. To gainmaximum expression of GalP, strains were grown overnight in minimalmedia with 15 mg/ml tetracycline, supplemented with L-histidine (80mg/ml) and thymine (20 mg/ml). For sugar transport and sugar-H1

symport experiments, requiring lower levels of expression, cells weregrown on rich media (2TY supplemented with 20 mM glycerol, 20 mg/mlthymine, and 15 mg/ml tetracycline).

Preparation of Inner Membranes Containing Over-expressed Pro-tein—The E. coli cells were disrupted by explosive decomposition in aFrench Press at 137.5 MPa, and the inner membranes were preparedessentially as described by Osborn et al. (44). This procedure yieldspredominantly inside-out vesicles (45).

Quantification of Over-expressed Protein—A sample of the mem-brane preparation (30 mg of protein) was subjected to SDS-polyacryl-amide gel electrophoresis. After staining with Coomassie Blue, gelswere scanned with a Molecular Dynamics 100A Computing Densitom-eter, and the proportion of GalP protein was measured. The levels ofexpression of the wild-type and mutant proteins were determined bycomparison of the intensities of the bands corresponding to the GalPproteins on Coomassie-stained SDS-polyacrylamide gels. The expres-sion of the Asn3943 Gln mutant protein was 107% that of the wild-typeprotein.

Protein Assays—The concentration of protein in the membrane prep-arations 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 ofglutamine (CAG) as follows. The single-stranded DNA template formutagenesis was prepared by subcloning a 2.46-kilobase pair AlwI-AlwI DNA fragment coding for the C-terminal half of GalP from plas-mid pPER3 (which confers elevated expression of GalP (2)) into bacte-riophage M13mp18. Mutagenesis was carried out with the AmershamCorp. in vitro mutagenesis kit using the mutagenic primer 59-GGCAATCCACTGGGTGGCAGTG-39 (the mismatches are underlined)complementary to the coding strand of galP. The base changes wereconfirmed by single-stranded dideoxynucleotide sequencing. A double-stranded 1.561-kilobase pair MluI-NdeI fragment containing the basechanges was isolated from the bacteriophage M13 construct and sub-stituted for the wild-type DNA in MluI-NdeI cut plasmid pPER3 to giveplasmid pTPM6 (Asn394 3 Gln). The substitution was confirmed bysubcloning the DNA containing the mutation back into bacteriophageM13mp18 and dideoxynucleotide sequencing. Double-stranded se-quencing of the entire mutant galP gene in the expression plasmidconfirmed that there were no other base changes in the galP genesequence.

Sugar Transport Measurements—The transport of radioisotope-la-beled sugars into intact cells was measured after the cells were ener-gized with 10 mM glycerol as described by Henderson et al. (43). Alltransport measurements were carried out on cells that had been grownon rich media producing levels of GalP expression less than 1% totalmembrane protein. The level of expression of the mutant GalP proteinwas very similar to that of wild type judging by Western blots (data notshown). The velocity of D-galactose uptake (with D-galactose at 50 mM)without the addition of inhibiting sugar did not vary greatly betweenexperiments showing little day to day variation (wild type 24.3 6 2.8nmol/mg/min (n 5 10) and Asn394 3 Gln 6.73 6 0.88 nmol/mg/min(n 5 10)).

Sugar-H1 Symport Measurements—D-Galactose-promoted pH changes(sugar-H1 symport) were measured using 3.3 mM sugar with energy-depleted 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 expressingwild-type and mutant GalP proteins were measured after the cells hadbeen aerated in the presence of glycerol (10 mM) and a range of sugarconcentrations for 3 min.

Permeabilization of the Cell Outer Membrane for Inhibition Studies—Cells were made permeable to cytochalasin B and forskolin by treat-ment with Tris-EDTA (48). Cells were grown for uptake experiments asdescribed above. After washing with the growth volume twice in 150 mM

KCl, 5 mM MES, pH 6.5, the cells were equilibrated in 200 mM Tris/HCl,pH 8.0, before the addition of an equal volume of 200 mM Tris/HCl, pH

8.0, 1 mM EDTA. After 30 min, 100 volumes of 150 mM KCl, 5 mM MES,pH 6.5, 10 mM MgSO4 were added and the cells harvested by centrifu-gation and resuspended in the same buffer.

Photoaffinity Labeling with 4-[3H]Cytochalasin B and IAPS-Forsko-lin—Ligands were incorporated into membrane proteins by irradiationwith ultraviolet light (13, 49). Inner membrane vesicles were preincu-bated with or without 500 mM D-galactose at 4 °C in photolabelingbuffer (50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.4)and transferred to quartz cuvettes containing 0.4 nM 3-125I-APS-for-skolin or 0.5 mM 4-[3H]cytochalasin B. The samples were flushed withargon to reduce free radical production and irradiated with ultravioletlight for 10 min. After irradiation, noncovalently bound ligand wasremoved by diluting the sample in photolabeling buffer containing 1%mercaptoethanol (for 3-125I-APS-forskolin) or unlabeled cytochalasin B(for 4-[3H]cytochalasin B) and centrifugation (130,000 3 g for 2 h at4 °C). The resulting membrane pellet was resuspended in 15 mM Tris/HCl, pH 7.5, and assayed for protein. The proteins were separated bySDS-polyacrylamide gel electrophoresis. The incorporation of 4-[3H]cy-tochalasin B was monitored by fluorography and the incorporation of3-125I-APS-forskolin by autoradiography.

Fluorescence Studies—Fluorescence spectra were measured in a Per-kin-Elmer LS50B spectrophotofluorimeter. The protein was excited ateither 280 or 297 nm and the fluorescence emission monitored between300 and 400 nm. Rapid reactions were followed using an AppliedPhotophysics (London, UK) spectrophotofluorimeter, operated at 20 °C,as described by Walmsley et al. (14, 17, 50). Fluorescence measure-ments 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 membraneswere used at a protein concentration of 200 mg/ml, in the above buffer.

Equilibrium Dialysis—Equilibrium dialysis was carried out as de-scribed by Walmsley et al. (50). The binding of 12-[3H]forskolin and4-[3H]cytochalasin B to inner membranes (1.0 mg/ml) containing GalPwas measured over the range of 0.05–80 mM ligand at 4 °C. Ratios ofbound to free ligand were calculated from the equilibrium distributionof the radiolabeled ligand and used to determine both the Kd and thenumber of ligand binding sites by an unweighted nonlinear least-squares fit of the data to an hyperbola using the Biosoft programUltrafit 2.1.

Statistical Analyses—The apparent Km values, with their standarddeviations, for transport of radiolabeled sugar were obtained by a least-squares fit of the unweighted initial rate data directly to a hyperbola

FIG. 2. Energized transport catalyzed by the Asn394 3 Glnmutant protein is mildly impaired. E. coli strain JM1100 express-ing the wild-type and mutant proteins were grown in 2 TY supple-mented with 20 mM glycerol until A680 5 0.6. The cells were harvestedand washed twice in the growth volume of 150 mM KCl, 5 mM MES, pH6.6, 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 ml of radiolabeled sugar to give theindicated concentration range. After 15 s, 2 ml was filtered rapidly andwashed four times in 2 ml of the above medium. The initial rates ofsugar uptake (nmol/min per dry mass) were calculated and displayed asa Lineweaver-Burk plot. The graph is a typical example of the uptake of1-[3H]D-galactose catalyzed by wild-type and mutant proteins. The datayielded Km values of 58.9 6 8.6 and 107.6 6 34.0 mM and Vmax values of65.4 6 2.3 and 28.5 6 2.2 nmol/mg/min for wild-type and mutantproteins, respectively. Similar experiments were carried out on theuptake of D-glucose and D-xylose, and the results are shown in Table I.

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using Biosoft program Ultrafit 2.1. Generally, rates of the quench in theintrinsic fluorescence of GalP, when it was mixed with the antibiotics ina stopped-flow apparatus, were fitted to an exponential function usingthe non-linear least-squares regression program supplied with the Ap-plied Photophysics SF.17MV stopped-flow equipment. The data sets, forthe concentration dependence of the rate of binding of the antibiotics,were analyzed by fitting to the appropriate equation using the non-linearleast-squares regression program SIGMA PLOT (Jandel Scientific).

RESULTS

Substitution of Asn394 by Gln Does Not Greatly Impair theSugar Transport Activity of GalP—The transport of D-galactoseby the wild-type GalP protein, under energized conditions, wascharacterized by a hyperbolic increase in the initial rate withincreasing concentrations of sugar. A least-squares fit yieldedapparent Km and Vmax values of 42.4–58.9 mM and 59.1–65.4nmol/mg/min (Fig. 2 and Table I). The Km and Vmax valuesquoted here and subsequently are apparent as it was notknown whether the co-substrate, H1, was at saturatingamounts. The substitution of Asn394 by Gln led to a 2-foldincrease in the Km for D-galactose and a 2-fold decrease in theVmax, compared with the wild type (Fig. 2 and Table I). Thedata indicated a relatively small (4.8-fold) reduction in thespecificity (as defined by Vmax/Km) of the mutant protein forD-galactose relative to the wild type. The uptake of 50 mM

D-galactose was accumulated by the mutant to 42-fold that ofthe external media, compared with 88-fold by the wild type.These data demonstrated that the mutant protein can catalyzeenergized transport and that the gross conformation of theprotein is not significantly impaired.

D-Glucose was transported into the mutant with a Vmax

1.5-fold higher than that of the wild-type protein, although theKm was 3.3-fold higher (Table I). The transport of D-xylose wascharacterized by a 4.3-fold increase in the Km and a 0.8-folddecrease in the Vmax compared with that of the wild type. TheVmax/Km values were 2.1-fold (D-glucose) and 5.3-fold lower(D-xylose) than that of the wild type.

The Asn394 3 Gln Mutant Protein Still Catalyzes D-Galac-tose-Proton Symport—The sugar-H1 proton symport activitiesof the wild-type and mutant proteins were assayed by monitor-ing D-galactose-promoted alkaline pH changes (data notshown), which are diagnostic of sugar-H1 symport (51, 52). Theextent of D-galactose-H1 symport catalyzed by the mutant pro-tein (5.19 6 2.7 nmol of H1 per mg (n 5 5)) was not signifi-cantly different from that of the wild-type protein (4.87 6 3.88nmol of H1 per mg (n 5 8)). However, the rate of D-galactoseH1-symport catalyzed by the mutant (1.16 6 0.41 nmol of H1

per mg (n 5 5)) was 2.8-fold lower that of the wild type (3.31 61.54 nmol of H1 per mg (n 5 8)). Again this implies that thegross conformation and transport competence of the mutantprotein are not seriously affected by the Asn394 3 Glnmutation.

D-Galactose Transport Catalyzed by GalP with the Asn394 3

Gln Mutation Was Inhibited by Forskolin but Not by Cytocha-lasin B—The abilities of cytochalasin B or forskolin to inhibitthe energized uptake of 50 mM D-galactose catalyzed by wild-type and mutant GalP into E. coli cells were investigated. Thecell walls were rendered permeable to the antibiotics by priortreatment with Tris-EDTA, pH 8.0 (see “Materials and Meth-ods”). In the case of the wild type, both cytochalasin B (80 mM)and forskolin (80 mM) inhibited the transporter, cytochalasin Bbeing a slightly more potent inhibitor (Fig. 3). However, thetransport catalyzed by the mutant protein was not significantlyinhibited by cytochalasin B, although it demonstrated amarked increase in sensitivity to inhibition by forskolin.

The Asn394 3 Gln Mutant Protein Was Photolabeled byIAPS-Forskolin but Not by Cytochalasin B—The wild-typeGalP protein was susceptible to photolabeling with 4-[3H]cy-tochalasin B and 3-125I-APS-forskolin, and the protein wasprotected against this photolabeling by the physiological sub-strate, D-galactose, but not by L-galactose, which is not a sub-strate (Fig. 4, lanes 2 and 3). This protection by substrateshowed that both cytochalasin B and IAPS-forskolin specifi-cally label the GalP protein (13–15).

FIG. 3. Energized uptake catalyzed by GalP with the Asn394 3Gln mutation is inhibited by forskolin but not cytochalasin B.Permeabilized cells from E. coli strain JM1100 expressing wild-type ormutant GalP were equilibrated with either 80 mM cytochalasin B, 80 mM

forskolin, or ethanol (solvent control) and 25 mM glycerol for 3 min. Theuptake of 50 mM 1-[3H]D-galactose was determined by taking a sample120 s after the addition of the sugar as described by Henderson et al.(43). The graph shows % transport compared with ethanol control.Wild-type samples with the addition of cytochalasin B are the mean of12 uptakes in three separate experiments; samples with the addition ofcytochalasin B are the mean of 6 uptakes in two separate experiments,and samples with the addition of forskolin are the mean of 3 samples ina single experiment. The mean values 6 the S.D. are shown. Energizeduptake catalyzed without the addition of antibiotic was 7.08 6 0.54nmol/mg/120 s21 for the wild type and 1.84 6 0.19 nmol/mg/120 s21 forthe Asn394 3 Gln mutant.

TABLE IThe kinetic parameters Km and Vmax for the transport of D-galactose, D-glucose, and D-xylose catalyzed by the wild-type and

Asn394 3 Gln mutant GalP proteinsInitial rates of the transport of the indicated radiolabeled sugar were measured over an appropriate range of concentrations, from about 0.5 Km

to 5 Km. The maximal rate of transport (Vmax) and the Km were determined by a least-squares fit of each data set to a Michaelis-Menten equation.

Parameter Wild-type Asn394 3 Gln

Km (D-galactose) 42.4–58.9 mM 107.6 6 3 mM

Vmax (D-galactose) 59.1–65.4 nmol/mg/min 28.5 6 2.2 nmol/mg/minVmax/Km (D-galactose) 1.4–1.1 nmol/mg/min/mM 0.26 nmol/mg/min/mM

Km (D-glucose) 10.2 6 3.5 mM 34.2 6 3.2 mM

Vmax (D-glucose) 15.6 6 1.4 nmol/mg/min 23.6 6 0.9 nmol/mg/minVmax/Km (D-glucose) 1.5 nmol/mg/min/mM 0.7 nmol/mg/min/mM

Km (D-xylose) 3800 6 600 mM 16500 6 7900 mM

Vmax (D-xylose) 297.8 6 25.5 nmol/mg/min 241 6 88.9 nmol/mg/minVmax/Km (D-xylose) 0.0768 nmol/mg/min/mM 0.0146 nmol/mg/min/mM

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The Asn3943 Gln mutant GalP protein was photolabeled by3-125I-APS-forskolin, and D-galactose, but not L-galactose, pro-tected the protein against the ligand; the extent of labeling andof protection were at levels similar to those of the wild-typeprotein (Fig. 4, B and D). However, no photolabeling ofthe mutant protein by cytochalasin B was detected (Fig. 4, Aand C).

The Asn394 3 Gln Mutant Protein Binds Forskolin but NotCytochalasin B—The equilibrium binding of the antibioticscytochalasin B and forskolin to GalP over-expressed in E. coli ischaracterized by linear Scatchard plots indicating a single spe-cies of high affinity binding sites; furthermore, one unlabeledantibiotic displaces the other labeled one in a simple competi-tive manner (2, 15). When the data were fitted to a singlehyperbolic function, by least-squares analysis (53), they re-vealed dissociation constants (Kd) for cytochalasin B binding of4.5 6 1.1 mM (n 5 10) and for forskolin binding of 1.36–1.43 mM

(2, 15). The binding sites were measured at 12.8 6 2.9 nmol/mgmembrane protein (n 5 10) and 7.2–8.1 nmol/mg membraneprotein for cytochalasin B and forskolin, respectively (2, 15).These values were compared with the amount of expressedGalP as determined by quantitative densitometry of SDS-poly-acrylamide gel electrophoresis gels (2, 54) and indicated a 1:1ratio for the binding of the antibiotics to the GalP protein.

To determine the concentration of the binding sites and the

Kd values for the binding of cytochalasin B and forskolin to themutant protein, equilibrium dialysis measurements were car-ried out with a range of antibiotic concentrations spanningbetween 0.05 and 80 mM (the maximum level of solubility). Inseparate experiments, the concentration of forskolin bindingsites was measured at 8.1 6 0.66 and 6.04 6 0.4 nmol/mgmembrane protein and is similar to that of the wild-type pro-tein. The Kd for forskolin binding was 1.28 6 0.34 and 2.16 60.69 mM (also similar to that of the wild-type GalP). In contrastto the binding of forskolin, no cytochalasin B binding could bedetected even when using a specific activity of 4-[3H]cytocha-lasin B 40-fold of that usually used. The horizontal Scatchardplot of the data set (Fig. 5) show that the small amount ofbound 4-[3H]cytochalasin B was not displaced by increasingamounts of cytochalasin B and is indicative of nonspecificbinding.

Measurement of the Steady-state Kinetic Parameters for theBinding of Antibiotics to the Asn394 3 Gln Mutant Protein—The binding of the antibiotics cytochalasin B and forskolin tothe over-expressed wild-type GalP protein was characterizedby a quench in the intrinsic protein fluorescence. This haspreviously allowed us to monitor the binding of these antibiot-ics by a combination of steady-state and stopped-flow spec-trofluorimetric techniques, providing a rigorous kinetic analy-sis of the binding process (14, 16).

FIG. 4. GalP with the Asn394 3 Gln mutation is photolabeled by forskolin but not by cytochalasin B. Inside-out membrane vesicleswere prepared from E. coli strain JM1100 expressing the wild-type and mutant protein using a French press as described under “Materials andMethods.” These were photolabeled with 4-[3H]cytochalasin B (0.5 mM) or 3-125I-APS-forskolin (0.4 nM) in the presence of either 500 mM D- orL-galactose. The photolabeled samples (30 mg) were separated on a 12.5% SDS-polyacrylamide gel and stained overnight with Coomassie Blue(cytochalasin B (A) and forskolin (B)) and subjected to fluorography (cytochalasin B (C)) and autoradiography (forskolin (D)). The characteristicappearance of the GalP protein is at 34–36 kDa. The lane order for both photolabeling experiments is as follows: lane 1, standards; lane 2, wildtype 1 D-galactose; lane 3, wild type 1 L-galactose; lane 4, Asn394 3 Gln 1 D-galactose; lane 5, Asn394 3 Gln 1 L-galactose.

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To assess the importance of Asn394 in the binding of antibi-otic, the intrinsic fluorescence of the mutant protein was ti-trated with antibiotic over a range of concentrations up to 80mM (Figs. 6 and 7). A fit of the data to a hyperbola yielded anoverall dissociation constant (Kd) for forskolin binding of 1.48 60.5 mM (n 5 3), similar to that of 1.83 6 0.56 mM determined forthe wild type (16). This is consistent with the equilibriumdialysis data (above), which showed similar Kd values for thebinding of forskolin to both wild-type and mutant proteins. Incontrast to the equilibrium dialysis and photolabeling experi-ments described above, some low affinity cytochalasin B bind-ing to the mutant protein was detected using the fluorescencemethod; however, this was shown to be with an affinity 37- and53-fold lower than the wild-type protein (Kd 41.4 and 58.5 mM)in two separate experiments.

The extent of the quenching of fluorescence by saturatingamounts of forskolin (10 mM) was compared between the wild-type and Asn394 3 Gln mutant proteins. Measurements weremade with lex at 280 and 297 (lem max 330–340 nm) to detectany differences due to light absorption by tyrosine and/or tryp-tophan residues and at two protein concentrations (50 and 100mg/ml) to detect any differences due to the light scatteringproperties of the membrane preparations; no differences in thelevel of quench was found at either wavelength or between thetwo protein concentrations in either preparation. A 1.4-foldincrease in the fluorescence quench of the mutant protein,relative to the wild type (wild type 8.09 6 0.32% (n 5 7); Asn394

3 Gln 11.36 6 0.88% (n 5 6)), was observed with lex 280 and1.8-fold increase with lex 297 (wild type 6.16 6 0.49% (n 5 5);Asn3943 Gln 11.06 6 0.14% (n 5 4)). A similar quantificationof the amount of fluorescence quench concomitant with thebinding of cytochalasin B to the mutant protein could not bemade as saturating amounts of ligand could not be obtained.

Measurement of the Transient Kinetic Parameters for theBinding of Antibiotics to the Asn394 3 Gln Mutant Protein—

The binding of either forskolin or cytochalasin B to the GalPprotein was previously analyzed by stopped-flow fluorescencespectroscopy in terms of the following three-step Model A(14, 16, 38).

ToL|;k1

k21

TiL|;k2 @A#

k22

Ti-antibioticL|;k3

k23

Ti/o-antibiotic

MODEL A

To and Ti are, respectively, the outward- and inward-facingforms of the unloaded transporter, and Ti-antibiotic is thetransporter-antibiotic complex. A further intermediate confor-mational change of the transporter-antibiotic complex was alsoproposed (Ti/o-antibiotic). Ti/o is possibly a state in which thetransporter is attempting to undergo reorientation and thus isa high affinity T-antibiotic complex.

For such a model, the dissociation constant of the transport-er-forskolin complex is given by Equation 1.

Kd 5K2~1 1 K1!

~1 1 1/K3!(Eq. 1)

where K1 5 k21/k1, K2 5 k22/k2, and K3 5 k23/k3.The Asn3943Gln GalP mutant protein was characterized by

a concentration-dependent linear increase in the rate of thefast phase of forskolin binding to the Ti conformation of theprotein (Fig. 6), indicating association (k2) and dissociation rateconstants (k22) of 1.51 6 0.1 mM21 s21 and 2.37 6 0.96 s21,respectively; the dissociation constant (K2 (k22/k2)) was calcu-lated to be 1.57 6 0.64 (Table II). The rates of association anddissociation of forskolin with the mutant were 4- and 3.6-foldslower, respectively, than for the wild type. Thus, the dissoci-ation constant (K2) of the mutant is almost identical to the wildtype, demonstrating that the affinity of the Ti or putativeinward-facing transporter of the mutant protein for forskolin isunchanged. As K2 and overall Kd values are similar for bothwild-type and mutant proteins there is no evidence to suggestchanges to either K1 or K3.

Cytochalasin B also bound to the mutant protein in a linear,concentration-dependent manner (Fig. 7), indicating an associ-ation rate constant of 0.67 6 0.28 mM21 s21. In previous workwith the wild-type GalP protein the dissociation rate constantfor cytochalasin B was measured by directly displacing ligand,bound to the transporter, by adding sugar substrate (49). Thetransport protein was previously equilibrated with 80 mM li-gand and mixed with 500 mM sugar in the stopped-flow appa-ratus to displace the bound ligand. The increase in proteinfluorescence resulting from displacement of the bound cytocha-lasin B by D-galactose could be fitted to a single exponentialfunction revealing a dissociation rate constant of 12.18 6 2.78s21. The dissociation constant K2 (k22/k2) for the Ti conforma-tion of the mutant protein was calculated as 18.1 6 8.78 mM.This value is 44-fold higher than that of the wild type and issimilar to the 37–53-fold decrease in the overall affinity (in-crease in overall Kd) for cytochalasin B.

The Binding of D-Galactose and D-Glucose to the InternalBinding Site Is Greatly Impaired in the Asn394 3 Gln Mutantof GalP—8-Anilino-1-naphthalene sulfonate (ANS) can be usedas a fluorescent probe to monitor conformational changes in theGalP protein induced by the binding of sugar (17). Transportedsugars such as D-galactose and D-glucose cause an enhance-ment in the ANS fluorescence; non-transported sugar ana-logues and antibiotics produce only a slight quench in the ANSfluorescence, but they can reverse the enhancement in fluores-cence induced by transported sugars. The increase in ANSfluorescence is attributable to the sugar-induced reorientation

FIG. 5. The binding of forskolin, but not of cytochalasin B, tothe Asn394 3 Gln mutant protein was detected by equilibriumdialysis. Inner membrane vesicles containing over-expressed Asn3943Gln mutant GalP was subjected to microdialysis against a range ofcytochalasin B or forskolin concentrations (0.05–80 mM), and the pro-portions of antibiotic bound to GalP and free in solution were measuredusing 12-[3H]forskolin or 4-[3H]cytochalasin B. The data were analyzedby a least-squares fit to a hyperbola from which the overall Kd forforskolin binding was calculated in two separate experiments to be1.28 6 0.34 and 2.16 6 0.69 mM and the number of binding sites to be8.1 6 0.66 and 6.04 6 0.4 nmol/mg membrane protein. The illustratedgraph shows the mean data of two experiments in the form of a “Scat-chard plot.” The horizontal line for cytochalasin B shows that the smallamount of bound 4-[3H]cytochalasin B was not displaced by increasingamounts of unlabeled antibiotic and is indicative of low level nonspecificbinding.

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of the transporter from an inward to an outward-facing confor-mation. Non-transported ligands are thought to reverse thefluorescence enhancement by recruiting the transporter to aninward-facing conformation. Before interpreting the effects ofthe Asn394 3 Gln mutation, we first considered the kineticmodel for the wild-type protein, which was derived from meas-urements of ANS fluorescence (17).

The rate of enhancement in ANS fluorescence produced byD-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 bindingof the sugar to the inward-facing conformation of the proteinand the slow phase to a subsequent conformational change.The rate of the fast phase increased hyperbolically with in-creasing D-galactose concentration indicative of the rapid bind-

ing of the transporter that was rate-limited by a slower confor-mational change at near saturating concentrations (seeEquation 2).

sugar 1 TiL|;KD-gal

sugar 2 TiL|;K2

sugar 2 To (Eq. 2)

Where Ti and To are inward- and outward-facing conforma-tional forms of the transporter, respectively; KD-gal is the dis-sociation constant for the inward-facing GalP-D-galactose com-plex and K2 5 k2/k22; k2 and k22 are rate constants for theconformational change subsequent to sugar binding and wereattributed to transporter reorientation.

The change in fast phase was fitted to the following hyper-bolic function as shown in Equation 3.

kobs 5k2@D-gal#

KD-gal 1 @D-gal#1 k 2 2 (Eq. 3)

Where kobs 5 the change in the fast rate; [D-gal] 5 D-galactoseconcentration; KD-gal 5 apparent dissociation constant for D-galactose. This yielded an apparent KD-gal of 7.22 6 1.49 mM

with minimum (k22) and maximum (k2) rates of 1.36 6 0.18 mM

and 4.06 6 0.16 respectively.The overall Kd for D-galactose binding was calculated at 1.8

mM from the measured values for KD-gal and K2 by the relation-ship shown in Equation 4.

Kd~overall! 5KD-gal

1 1 K2(Eq. 4)

However, it was also measured from the total fluorescencechange since the sum of the fast and slow fluorescence phasesalso exhibited hyperbolic dependence upon the D-galactose con-centration with an overall Kd of 0.66 6 0.033 mM. As thecalculated value for the overall Kd is greater than the measuredvalue, a further conformational change following the formationof To-D-gal causing additional tightening of the binding of D-galactose was proposed. The calculated Kd for the binding ofglucose (0.33 mM) was also greater than the measured value(0.18 mM). These results led to the suggestion that the kineticscheme shown above be expanded to give Equation 5.

FIG. 6. Fluorescence titration demonstrates that the Asn394 3Gln mutant binds forskolin with an affinity similar to wild type.A shows the concentration dependence of the total quench in the Asn394

3 Gln and wild-type proteins induced by the binding of forskolin, fittedby least-squares to a hyperbolic equation. Three separate experimentsyielded an overall dissociation constant (Kd) of 1.48 6 0.5 (n 5 3)compared with 1.83 6 0.56 (n 5 3) of the wild type. B shows theconcentration dependence of the observed rate of binding of forskolin tothe Asn394 3 Gln mutant and wild-type protein. Linear regressionanalysis of the data shown yielded values of 1.51 6 0.099 mM21 s21

(Asn394 3 Gln mutant) and 6.16 6 0.511 mM21 s21 (wild type) for theassociation rate constant (k2); and 2.37 6 0.962 s21 (Asn394 3 Glnmutant) and 10.43 6 3.23 s21 (wild type) for the dissociation rateconstant (k22). These values yield 1.57 6 0.64 mM (Asn394 3 Glnmutant) and 1.71 6 0.55 mM (wild type) for the apparent dissociationconstant (K2) of forskolin binding.

TABLE IIKinetic parameters for the binding of antibiotic to the

Asn3943 Gln mutant

Parameter Wild-type Asn394 3 Gln

Kda (forskolin) 1.83 6 0.56 mM 1.48 6 0.5

Kdb (forskolin) 1.36–1.43 mM 1.28–2.61

k22c (forskolin) 8.62 6 2.49 s21 2.37 6 0.96

k2c (forskolin) 5.95 6 0.36 mM21 z s21 1.51 6 0.1

K2d (forskolin) 1.45 6 0.43 mM 1.57 6 0.64

DFmaxe (forskolin) 6.16 6 0.49% 11.06 6 0.14

Kda (cytochalasin B) 1.1 6 0.19 41.4–58.5

k22f (cytochalasin B) 2.5 6 0.2 s21 12.18 6 2.75

k2c (cytochalasin B) 6.1 6 0.14 mM21 z s21 0.67 6 0.28

K2d (cytochalasin B) 0.41 6 0.03 mM 18.1 6 8.78a The overall dissociation constants (Kd) were determined by titration

of the protein fluorescence (DFmax).b The overall dissociation constants (Kd) were determined by equilib-

rium dialysis.c Association and dissociation rate constants (k2 and k22) were deter-

mined from the linear plot of the observed rate of antibiotic bindingversus antibiotic concentration.

d The dissociation constant for antibiotic binding to the inward-facingantibiotic site (K2) was calculated from the measured association (k2)and dissociation (k22) rate constants (k2/k22).

e DFmax values were measured at steady state with saturatingamounts of forskolin using right-side-out inner membrane vesicles withlex 297 and lem 335.

f Dissociation rate constant was measured by the displacement ofcytochalasin B by D-galactose.

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sugar 1 TiL|;KD2gal

sugar 2TiL|;K2

sugar 2 ToL|;K3

sugar 2 To* (Eq. 5)

Where To* is an outward-facing conformation, slightly different

to that of To.Hence the Kd would be as shown in Equation 6.

Kd 5KD-gal

1 1 K2~1 1 K3!(Eq. 6)

In contrast to the wild-type protein, binding of D-galactoseand D-glucose to the mutant protein was characterized by amonophasic increase in the ANS fluorescence with time. Therate was apparently independent of sugar concentration, and itwas not possible to determine rate constants, k2 and k22, andthe dissociation constant, KD-gal. It is probable that k2 and k22

are very similar. The total fluorescence enhancement, which

was 8.5 times lower in the case of D-galactose and 3.6 timeslower in the case of D-glucose, did, however, exhibit a hyper-bolic dependence upon the concentration of sugar (Fig. 8). Theconcentration dependence of the total fluorescence enhance-ment for D-galactose and D-glucose fitted to a hyperbolic func-tion and from three separate experiments yielded overall Kd

values of 42.3 6 11.8 for D-galactose and 29.4 6 3.5 for D-glucose. These Kd values are a 64.1- and 163.3-fold increase ofthe wild type for D-galactose and D-glucose, respectively. As itwas possible only to measure the overall Kd for sugar binding,which encompasses the initial binding event to the inward-facing conformation (Ti) and the translocation of sugar acrossthe membrane (see three-step kinetic Model A above), it cannotbe ruled out that the mutation has affected any of the subse-quent reorientation steps. However, the energized transport ofD-glucose and D-galactose catalyzed by the mutant protein intowhole cells is not severely affected, implying that the sugartranslocation mechanism remains intact and that it is thebinding of sugar to the internal binding site that is predomi-nantly impaired.

Substrate Specificity of the Asn394 3 Gln Mutant ofGalP—To determine the substrate specificity of the Asn394 3

FIG. 7. Fluorescence titration demonstrates that the Asn394 3Gln mutant binds cytochalasin B with much reduced affinity. Ashows the concentration dependence of the total quench in the Asn3943Gln and wild-type proteins induced by the binding of cytochalasin B,fitted by least-squares to a hyperbolic equation. Repeated experimentsyielded an overall dissociation constant (Kd) of 41.4–58.5 mM for theAsn3943Gln protein compared with 1.1 6 0.19 (n 5 4) for the wild type.B shows the concentration dependence of the observed rate of binding ofcytochalasin B to the Asn394 3 Gln mutant and wild-type protein.Linear regression analysis of the data shown yielded values of 0.673 60.282 mM21 s21 (Asn3943 Gln mutant) and 6.14 6 0.102 mM21 s21 (wildtype) for the association rate constant (k2); 12.18 6 2.78 s21 (Asn394 3Gln mutant) and 2.51 6 0.65 s21 (wild type) for the dissociation rateconstant (k22); with 18.1 6 8.78 mM (Asn3943 Gln mutant) and 0.41 60.106 mM (wild type) for the apparent dissociation constant (K2).

FIG. 8. The dissociation constant for sugar binding and trans-location is drastically altered by the Asn3943 Gln mutation. Thedata show the ANS fluorescence enhancement as a function of theconcentration of D-glucose (A) and D-galactose (B) fitted by least squaresto a hyperbolic equation. Repeated experiments yielded an overall dis-sociation constant (Kd) of 29.4 6 3.5 (n 5 4) (Asn3943 Gln) and 0.18 60.01 mM (wild type) for D-glucose and 42.3 6 11.8 mM (n 5 3) (Asn3943Gln) and 0.66 6 0.16 mM (wild type) for D-galactose.

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Gln mutant protein, an extensive kinetic analysis was carriedout in which increasing concentrations of the unlabeled sugarunder investigation were used to inhibit labeled 1-[3H]D-galac-tose transport over a range of concentrations of both sugars.

Initial velocities of D-galactose uptake catalyzed by the wild-type and mutant proteins into E. coli were measured. The 1/vagainst [I] plots (Dixon plots) for D-galactose were linear, asexpected for simple hyperbolic binding, in the presence of un-labeled D-galactose, 2-deoxy-D-glucose, D-talose, 6-deoxy-D-glu-cose, 2-deoxy-D-galactose and D-mannose. At higher concentra-tions of D-xylose, 6-deoxy-D-galactose (wild type only),D-glucose, and L-arabinose the Dixon plots were non-linear anddeviated systematically from hyperbolic binding (data notshown). However, the data points at the lower concentrations ofthese inhibiting substrates did display linearity, and extrapo-lated estimates of Ki were determined. It was reported previ-ously (13) that the kinetics of transport of D-glucose, 2-deoxy-D-glucose, and 6-deoxy-D-galactose transport by GalP were non-hyperbolic. In order that changes in substrate specificity of themutant protein could be readily detected, a ratio of mutant Ki

to wild-type Ki was calculated, which was then normalized bydividing with the ratio for D-galactose (Table III). The recogni-tion by the mutant protein of most of the sugar substrates wasonly altered from that of the wild type between 0.5–2.0-fold,indicating little change in sugar selectivity. Surprisingly, thespecificity for D-xylose was seven times lower with the mutantprotein compared with the wild type. There is no obvious,simplistic interpretation of this effect such as an altered sub-strate recognition in terms of a single H-bonded sugar-proteininteraction. Clearly the mutation of the Asn394 residue to Glnhas, in terms of D-xylose, selectively affected the substratespecificity of the GalP protein.

DISCUSSION AND CONCLUSIONS

An alignment of the amino acid sequences of those proteinsthat are homologous to GalP, together with a knowledge of theability of these transporters to bind cytochalasin B, suggestedthat the Asn394 residue might be involved in the recognition ofcytochalasin B as a part of the cytochalasin B binding site orpocket. For the purposes of this work we will define the bindingsite as comprising those amino acids that bind directly with theantibiotic, and the binding pocket is defined as amino acids inthe immediate environment of the antibiotic that do not nec-essarily bind directly to it. To investigate the involvement ofAsn394, the residue was altered by mutagenesis to Gln.

Inward D-galactose transport catalyzed by the mutant pro-tein, as defined by the parameters Km and Vmax, was relativelyunimpaired, implying that the gross conformation of the pro-

tein was intact and the transport mechanism conserved. Sim-ilarly, the mutant protein was able to catalyze sugar protonsymport, to an extent similar to wild type albeit at a 2.8-foldlower rate, indicating that coupling between proton and sugartransport was maintained.

Previous equilibrium binding experiments on the wild-typeprotein revealed mutual competition between cytochalasin Band forskolin for the antibiotic binding site (16). It was there-fore of interest to investigate the effects of the Asn394 3 Glnsubstitution on the interaction of GalP with both antibiotics.The mutant protein was photolabeled by IAPS-forskolin atlevels similar to that of the wild-type protein, and the reactionwas protected by D-galactose. These data showed that Asn394 isnot essential for photolabeling by forskolin. However, it cannotbe ruled out that Asn394 of wild-type GalP is the photolabeledamino acid residue and that Gln394 is being photolabeled in themutant protein. Positive identification must wait for the directsequencing of protein fragments generated from photolabeledtransporter. A Scatchard analysis demonstrated that the over-all affinity for forskolin binding to the mutant protein and thenumber of forskolin binding sites were almost identical to thoseof the wild-type protein; this unchanged overall affinity of themutant protein for forskolin was confirmed by the titration ofthe protein’s fluorescence. D-Galactose transport catalyzed bythe mutant protein demonstrated a marked increase in sensi-tivity to inhibition by forskolin compared with the wild type(wild type 58.0 6 2.9% and Asn3943 Gln 7.3 6 2.9% comparedwith the ethanol control). This increase probably reflects adecrease in the level of competition by D-galactose. The maxi-mal fluorescence quench induced by forskolin binding was 1.8-fold greater with the mutant protein than the wild type (lex

297; lem max 330–340 nm), although the amount of GalP wasthe same. This indicates that the substitution of Asn394 by Glnaltered the environment of one or more tryptophan residues.Characterization of a Trp395 3 Phe mutant GalP protein indi-cated that Trp395 is in a conformationally active region of theprotein and that this residue is the main reporter of forskolinbinding in fluorescence quenching experiments (38). The in-crease in the maximal fluorescence quench due to the bindingof forskolin to the Asn3943 Gln mutant can be rationalized bysuggesting that the presence of Gln394 causes a localized con-formational change altering the environment of the neighbor-ing Trp395 residue. Although the overall affinity for forskolinbinding to the mutant protein is unaffected, transient fluores-cence measurements showed that the rates of association (k2)and dissociation (k22) to the inward-facing conformation of theprotein (Ti) are reduced (3.9- and 3.6-fold for k2 and k22, re-

TABLE IIISugar recognition by the wild-type and Asn394 3 Gln mutant GalP proteins

Ki values for the given sugar were determined by measuring the initial velocity of energized 1-[3H]D-galactose uptake catalyzed by the wild-typeand mutant proteins into host E. coli strain JM1100 in the presence of an appropriate range of inhibitory sugar concentrations from about 0.5 Kito 10 Ki. The Ki values were calculated by a least squares fit to a hyperbola. A ratio of the wild type to mutant Ki values for each inhibitory sugaris shown. To aid comparisons of the change in substrate specificity of the mutant protein, this ratio has been normalized to that of D-galactose.

Sugar Wild-type Ki Asn394 3 Glu KiAsn394 Gln 3 (Ki)

Wild-type (Ki)Comparison

normalized toD-Galactose

mM mM

D-Glucose 34.1 6 11 84.0 6 24 2.46 1.17D-Galactose 103.6 6 12.6 306 6 73 2.9 12-Deoxy-D-glucose 236 6 79 322 6 98 1.3 0.45D-Talose 352 6 42.2 1392 6 136 3.95 1.366-Deoxy-D-glucose 413 6 144 662 6 150 1.6 0.556-Deoxy-D-galactose 513 6 80 3853 6 622 7.5 2.12-Deoxy-D-galactose 630 6 201 1844 6 529 2.9 1D-Mannose 789 6 160 4944 6 1697 6.26 2D-Xylose 1271 6 330 28128 6 545 22 7.6L-Arabinose 6649 6 453 47193 6 13637 7 2.4

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spectively). The affinity for the initial interaction with forskolinto the inward-facing conformation of the protein (Ti), as definedby the dissociation constant K2 (k22/k2), was unchanged. As K2

and overall Kd values are similar for both wild-type and mutantproteins, there is no evidence to suggest changes to either K1

or K3.In stark contrast to the binding of forskolin, cytochalasin B

was shown, in fluorescence quenching experiments, to bind tothe mutant protein with an overall affinity 37–53-fold lowerthan that of the wild-type protein. The displacement of cytocha-lasin B from the mutant protein by sugar substrate providedcorroborating evidence of ligand binding. This low affinity cy-tochalasin B binding was not detectable at all in photolabelingor equilibrium dialysis experiments, most likely due to aninsufficient concentration or specific activity of the radiolabeledcytochalasin B.

Transient fluorescence measurements showed that the affin-ity for the initial interaction with cytochalasin B to the inward-facing conformation of the protein (K2, as defined by k22/k2)was also much lower than wild type (40-fold lower). The reduc-tion in overall affinity for cytochalasin B cannot be attributedto stabilization of the To (outward-facing) conformation of themutant transporter, otherwise a similar effect should be ob-served with forskolin binding; thus, it is most likely to beentirely due to a reduction in affinity of the Ti conformation.Cytochalasin B was shown to have a rate of association to themutant protein (k2) lower than that of the wild type (9-fold) andas such is similar to forskolin. However, in direct contrast withthe rate of forskolin dissociation, that of cytochalasin B fromthe mutant protein is higher (4.9-fold) than that of the wildtype.

Possible explanations for these data are that the amino acidsubstitution has 1) altered the shape of the forskolin bindingpocket making it more difficult for the antibiotic to bind, butonce bound it is held within the pocket for longer, and (2)disrupted a single H-bonded cytochalasin B-protein interactionresulting in a reduction of overall affinity for the ligand. Thereduction in affinity for cytochalasin B and alteration of for-skolin binding provides evidence that the cytochalasin B andforskolin binding sites are closely associated. We speculate thatAsn394 forms part of the cytochalasin B binding site and part ofthe forskolin binding pocket. However, the possibility that thedisruption in cytochalasin B binding is due to steric hindranceby the larger Gln residue cannot be discounted.

In contrast to the energized transport of sugar, which isrelatively unimpaired, ANS fluorescence measurements showthat the overall Kd describing the binding of D-galactose andD-glucose to the inward-facing conformation of the mutant pro-tein and their subsequent translocation across the membranewere drastically higher than that for the wild-type protein(64.1-fold for D-galactose and 163.3-fold for D-glucose). The factthat energized transport catalyzed by the mutant transport isrelatively unimpaired suggests that the translocation mecha-nism is intact. It is therefore most likely that the decrease inoverall affinity is largely due to loss of affinity for these sugarsat the inward-facing binding site.

The substitution of the Asn394 residue by Gln has, at least, atthe primary structural level made the GalP protein more likeXylE. Such single residue differences may be the means bywhich proteins are able to discriminate between different sub-strates, e.g. between hexoses and pentoses. The sugar specific-ity of the wild-type GalP protein has been extensively investi-gated (2, 13, 15, 17), which showed the importance of the C-3hydroxyl and C-6 on the pyranose ring for hexose recognitionby GalP and the lack of importance of the C-2 and C-4 hydroxylgroups. These observations parallel the requirements for sugar

recognition by the passive glucose transporters of humanerythrocytes (GLUT1) and rat adipocytes (GLUT4) (11, 12),confirming the functional similarity of the bacterial and mam-malian transporters. The substrate specificity study carriedout for energized uptake of sugars in this work did not reveal ageneral role for the Asn394 residue in the recognition of exter-nal sugar substrates, but, surprisingly, the specificity for D-xylose was significantly reduced (7.6-fold compared with thewild type); there is no obvious, simplistic interpretation of thiseffect, such as an altered substrate recognition in terms of asingle H-bonded sugar-protein interaction. Overall, these ob-servations support the other data that indicate Asn394 is part ofthe internal binding site for sugars.

Cytochalasin B has been shown by kinetic analysis to com-pete with D-glucose for binding to the inward-facing conforma-tion of the GLUT1 protein (55). Our data, which demonstratethat the disruption of sugar binding to the inward-facing con-formation of GalP (as opposed to the outward-facing conforma-tion) is coupled to the disruption of cytochalasin B binding bythe Asn394 3 Gln mutation, provide further evidence of thefunctional and structural similarity of the GLUT1 and GalPproteins. These data contribute to the ongoing debate as towhether cytochalasin B and sugar bind to the same site of theinhibitor susceptible proteins and suggest, at least with GalP,that this is the case.

An Asn4153 Asp mutant of the GLUT1 transporter has beenisolated (56). Asn415 is conserved in all the GalP, AraE, XylE,and GLUT transporters and is the 4th residue after Asn394 inGalP. Characterization of the Asn415 3 Asp mutant GLUT1protein showed 40% less cytochalasin B photolabeling althoughthe inhibition of labeling by ethylene glucose, which preferen-tially binds to the outer glucose binding site (57), inhibitedcytochalasin B similar to the wild type. This led to the sugges-tion that the Asn415 residue is likely to reside close to the innerglucose and cytochalasin B binding sites of GLUT1. It is inter-esting to note that to make a complete turn of an a-helix, thepolypeptide backbone must traverse 3.6 amino acids. Thusamino acids three or four amino acids apart (as in the case ofAsn412 (aligned with Asn394 of GalP) and Asn415 of GLUT1)would be expected to project from the same side of an a-helix.It has also been shown (38) that the affinity for the initialinteraction of cytochalasin B to a Trp395 3 Phe mutant asdescribed by the apparent dissociation constant (K2) is 43-foldlower than that for the wild type while the K2 for forskolinbinding to the mutant is similar to wild type. These datasuggest that the residue adjacent to Asn394 (Trp395) is alsoassociated with the cytochalasin B but not the forskolin bindingsite. Interestingly, the affinity for D-galactose of the inward-facing binding site of the Trp395 3 Phe mutant is similar towild type and is therefore unlikely to be involved in D-galactosebinding (38). It is by the collation of such data from manymutants that structural and mechanistic models for these re-lated sugar transporters will begin to emerge, and the bindingsites of sugar and antibiotic will be determined.

Acknowledgments— We are also grateful to J. O’Reilly for isolatingthe inner membrane preparations from E. coli and D. Ashworth foroligonucleotide synthesis and DNA sequencing. We are indebted to Dr.M. F. Shanahan for the kind gift of 3-125I-APS-forskolin.

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Cytochalasin B Insensitive Mutant of GalP 15199

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Terence P. McDonald, Adrian R. Walmsley and Peter J. F. Hendersonand Sugar

Is Associated with the Internal Binding Site for Cytochalasin BEscherichia colifrom Symport Protein (GalP)+Asparagine 394 in Putative Helix 11 of the Galactose-H

doi: 10.1074/jbc.272.24.151891997, 272:15189-15199.J. Biol. Chem. 

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