From the Howard Hughes Medical Institute, Departments
of Physiology and Microbiology & Molecular Genetics and the Molecular
Biology Institute, UCLA, Los Angeles, California 90095-1662 and
the § Institute of Organic and Medicinal Chemistry,
University of Pécs, H-7643 Pécs, Hungary
Received for publication, November 6, 2002, and in revised form, December 4, 2002
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ABSTRACT |
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Affinity inactivators are useful for probing
catalytic mechanisms. Here we describe the synthesis and properties of
methanethiosulfonyl (MTS) galactose or glucose derivatives with respect
to a well studied membrane transport protein, the lactose permease of
Escherichia coli. The MTS-galactose derivatives behave as
affinity inactivators of a functional mutant with
Ala122 The lactose permease
(LacY)1 of Escherichia
coli transduces the free energy stored in an electrochemical
H+ gradient into a concentration gradient of
D-galactopyranosides and vice versa
(galactoside/H+ symport) (1-4). LacY is a
12-transmembrane-helix bundle with the N and C termini on the
cytoplasmic face of the membrane (5-7). Several lines of evidence
indicate that it is both functionally (8) and structurally a monomer
(9-11). Analysis of an extensive library of mutants, particularly Cys
replacement mutants (12), with a battery of site-directed biophysical
and biochemical techniques has led to the formulation of a teriary
structure model (49) that includes tilts as well as a working model for
the transport mechanism (13).
LacY is selective for disaccharides containing a
D-galactopyranosyl ring as well as D-galactose
(14-16), but it has no affinity for D-glucose or
D-glucopyranosides (16, 17). The specificity of LacY is
directed toward the galactosyl moiety of the substrate, and the C-4 OH
is most important for ligand binding (16, 18). The major determinants
for substrate binding are located at the interface between helices IV,
V (19-24), and VIII (50). Alkylation of single-Cys122 LacY
(helix IV) with N-ethylmaleimide (NEM) abolishes lactose transport as well as ligand binding. Moreover, lactose, melibiose, and
Affinity labeling is based upon specific recognition of an inactivating
ligand at the binding site. Because Cys148 (helix V), which
is in close proximity to Ala122, makes direct contact with
the galactopyranosyl moiety of the substrate, numerous attempts to
develop an affinity reagent specific for this side chain have failed.
On the other hand, Ala122 abuts the non-galactosyl moiety
of disaccharide substrates, and alkyation of a Cys residue at this
position blocks binding of disaccharides with no effect on galactose
transport or binding. Therefore, methanethiosulfonyl (MTS) galactoside
with an ethylene linkage between the galactopyranoside ring and MTS and
MTS-5-galactose with a carbamide linkage were synthesized as potential
affinity reagents for the single-Cys A122C mutant in LacY with the
corresponding glucosides as controls.
Reagents
MTS-galactoside and MTS-glucosideCys in a background otherwise devoid of Cys
residues. A proton electrochemical gradient
(
Cys mutation
is combined with a mutation (Cys154
Gly) that blocks
transport but increases binding affinity, discrimination between the
MTS-galactose and -glucose derivatives is abolished, and
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranosyl
1-thio-
-D-galactopyranoside (TDG) protect against NEM
inactivation of lactose transport as well as labeling of
Cys122 by [14C]NEM. Remarkably, however,
D-galactose transport is essentially unaffected by NEM, and
the monosaccharide affords no protection against inactivation of
lactose transport or labeling of Cys122 by NEM.
Consistently, competitive inhibition of galactose transport by lactose,
melibiose, or TDG is drastically reduced after alkylation by NEM,
whereas inhibition by unlabeled galactose is unaffected. Finally,
replacement of Ala122 with Phe or Tyr has the same effect
as alkylation of single-Cys mutant A122C with NEM. The results
demonstrate that Ala122 is in close proximity to the
nongalactosyl portion of ligand (24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MTS-galactoside
and MTS-glucoside were synthesized as shown in the synthetic scheme
(Scheme 1). Reagents and conditions were
as follows: (i) bromoethanol 1.25 eq,
BF3Et2O 5.1 eq,
CH2Cl2, 0 °C
room temperature, 24 h, (53-61%); (ii) NaOMe 0.1 eq, MeOH, 4 h, and
then Dowex 50-W (H+),
NaSSO2CH3 (1.3 eq),
N,N-dimethylformamide, 50 °C, 16 h
(36-42%).
View larger version (6K):
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Scheme 1.
Synthetic scheme.
Melting points were determined with a Boetius micro melting point apparatus and are uncorrected. A synthesis of compounds 3a and 3b was published previously (25). All of the chemical and physical data were identical to those published previously. All of the chemicals were purchased from Sigma, and the NaSSO2CH3 was synthesized as described by Mintel and Westley (26). Preparative flash column chromatography was performed on Merck Kieselgel 60 (0.040-0.063 mm). Qualitative TLC was carried out on commercially prepared plates (20 DC-Alufolien 5 × 7.5 cm) coated with Merck Kieselgel 60 F254 and visualization was achieved by heating the developed plate.
2-(2,3,4,6-Tetra-O-acetyl--D-galactopyranosyl)ethyl
Bromide (2a) and
2-(2,3,4,6-Tetra-O-acetyl-
-D-glucopyranosyl)ethyl
Bromide (2b)--
To a stirred solution of
-D-galactose-pentaacetate (1a) or
-D-glucose-pentaacetate (1b) (3.9 g, 10.0 mmol) and bromoethanol (0.88 ml, 12.4 mmol),
BF3·Et2O (6.5 ml, 51.0 mmol) in
CH2Cl2 (20 ml) was added dropwise at 0 °C,
and the mixture was allowed to warm to room temperature and stirred for
an additional 24 h. The mixture was poured over ice-water
(20 ml) and separated, and the aqueous phase was washed with
CH2Cl2 (3 × 10 ml); the combined organic
phase was washed with 5% aqueous NaHCO3 (10 ml) and water
(10 ml), dried (MgSO4), filtered, and evaporated; and the
residue was purified by flash column chromatography (hexane/EtOAc) to
give the title compounds 2a (2.77 g (61%), melting point 108-110 °C, Rf 0.35 (hexane/EtOAc, 2:1)) and
2b (2.41 g (53%) 113-115 °C, Rf
0.32 (hexane/EtOAc, 2:1)).
2-(-D-Galactopyranosyl)ethyl Methanethiosulfonate
(3a) and 2-(
-D-Glucopyranosyl)ethyl
Methanethiosulfonate (3b)--
To a stirred solution of
compound 2a or 2b (455 mg, 1.0 mmol) in MeOH (10 ml), a freshly made 0.1 M NaOMe solution in MeOH (1 ml) was
added, and the mixture was stirred for further 4 h. The reaction
mixture was then passed through a Dowex 50-W (H+) (2.5 × 2-cm plug, eluant MeOH), and the solvent was removed in
vacuo to give an oil 215 mg (74%) for 3a and 200 mg (69%) for 3b. This product was immediately dissolved in N,N-dimethylformamide (10 ml);
NaSSO2CH3 (134 mg, 1.0 mmol) was added, and the
mixture was heated at 50 °C for 15 h. Then,
N,N-dimethylformamide was evaporated, and the
residue was purified by flash column chromatography (EtOAc/MeOH) to
give the title compounds as white thick oils that solidified upon
cooling (3a, 133 mg (42%), Rf 0.38 (EtOAc/MeOH, 4:1); 3b, 114 mg (36%),
Rf 0.48 (EtOAc/MeOH, 4:1)).
MTS-5-galactose and -glucoseMTS-5-galactose was
custom synthesized by Toronto Research Chemicals (Toronto, Canada), and
MTS-5-glucose was purchased from the same source.
Other Reagents-- [1-14C]Lactose was obtained from Amersham Biosciences. N-([14C]ethyl)Maleimide was purchased from PerkinElmer Life Sciences. [3H]NPG was the generous gift of Gérard Leblanc (Villefranche-sur-mer, France). Immobilized monomeric avidin was from Pierce, and all unlabeled sugars were obtained from Sigma. All other materials were reagent grade and were obtained from commercial sources.
Construction of LacY Mutants
All mutants were constructed in a cassette lacY gene encoding functional LacY devoid of Cys residues with a C-terminal biotin acceptor domain (27, 28). Plasmids pKR35/A122C/C148S and pKR35/A122C/C148A have been described (24, 29). pKR35/A122C/C148D and pKR35/A122C/C148A/C154G were generated by oligonucleotide-mediated, site-directed mutagenesis using two-step PCR with pKR35/A122C/C148A as template.
Growth of Cells
E. coli T184
(lacI+O+ZY
(A)
rpsL,met
,thr
,recA,hsdM,hsdR/F',lacIqO+ZD118(Y+A+))
containing given mutants was grown in Luria-Bertani broth with 100 mg/liter of ampicillin. Overnight cultures were diluted 10-fold and
allowed to grow for 2 h at 37 °C before induction with 1 mM isopropyl 1-thio-
-D-galactopyranoside.
After additional growth for 2-3 h at 37 °C, cells were harvested by centrifugation.
Preparation of Right-Side-Out Vesicles
Right-side-out (RSO) membrane vesicles were prepared by osmotic
lysis as described (30, 31), suspended in 100 mM potassium Pi (pH 7.5)/10 mM MgSO4 at a
protein concentration of about 12 mg/ml, frozen in liquid
N2, and stored at 80 °C until use.
Inactivation by MTS Sugars
RSO vesicles at a protein concentration of 3 mg/ml were incubated at 24 °C with a given MTS sugar in the absence or presence of 20 mM ascorbate and 0.2 mM phenazine methosulfate (PMS) under oxygen (32, 33). Reactions (0.25 ml, total volume) were quenched by the addition of 1.75 ml of ice-cold 100 mM potassium Pi (pH 7.5)/10 mM MgSO4 at the indicated times and immediately centrifuged. The pellet was washed with 2.0 ml of the same buffer, centrifuged, and resuspended in 0.25 ml of 100 mM potassium Pi (pH 7.5)/10 mM MgSO4.
Transport Assays
Lactose transport with [1-14C]lactose (10 mCi/mmol) at a final concentration of 0.4 mM was carried out in the presence of 20 mM ascorbate and 0.2 mM PMS under oxygen as described previously (33). For mutant A122C/C148D, measurements of the rate of lactose transport were studied with vesicles washed in 100 mM potassium Pi/10 mM MgSO4 (pH 5.5), because this mutant exhibits a higher rate of transport at acidic pH (34).
NEM Labeling
The Kd for TDG binding was determined in situ by alkylation of given mutants with [14C]NEM in the absence or presence of given concentrations of TDG (35, 36).
To determine the reaction of Cys122 with MTS sugars, vesicles were incubated with an MTS sugar at a given concentration for indicated periods of time, diluted and washed with 100 mM potassium Pi (pH 7.5)/10 mM MgSO4 as described above, and concentrated to a protein concentration of 15 mg/ml. [14C]NEM (40 mCi/mmol; 0.5 mM final concentration) was added for 30 min, and the reaction was quenched by adding 20 mM dithiothreitol. The samples were then treated as described (29, 35).
Flow Dialysis
Binding of [3H]NPG to RSO vesicles containing given LacY mutants was measured by flow dialysis as described (37).
Determination of Methanthiosulfonate Concentration
The concentration of MTS reagents was determined by measuring
the disulfide exchange among 5,5-dithio-bis-(2-nitrobenzoic acid), dithiothreitol, and MTS (38).
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RESULTS |
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MTS Sugars--
MTS-galactoside and MTS-glucoside (Fig.
1) were synthesized as described under
"Experimental Procedures." MTS-5-galactose and MTS-5-glucose
were custom-synthesized by Toronto Research Chemicals.
|
Inactivation of LacY by MTS Sugars--
When RSO membrane vesicles
expressing single-Cys mutant A122C with Cys148 replaced by
Ala were treated with given concentrations of MTS-galactoside or
MTS-glucoside for 10 min and initial rates of lactose transport were
measured, EC50 values (the concentration at which
half-maximal inhibition is observed) approximating 36 and 130 µM, respectively, were obtained (Fig.
2A, Table I). However, it is
noteworthy that at low concentrations of
MTS-galactoside, a significant percentage of the reagent reacts with Cys residues unassociated with LacY, making
it difficult to determine a true
EC50.2
MTS-5-galactose, which is more hydrophilic because of the carbamide linkage (Fig. 1), exhibits an EC50 of about 151 µM, whereas MTS-5-glucose has an EC50 well in
excess of 800 µM (Fig. 2B).
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To demonstrate that the differential effect of MTS-galactoside and MTS-glucoside on transport is due specifically to the reaction with the Cys residue at position 122, blockade of [14C]NEM labeling by the MTS sugars was studied (Fig. 2C). RSO vesicles were treated under the same conditions as described for transport and then labeled with NEM. Prior to treatment with MTS-galactoside, Cys122 reacted well with NEM, and treatment with MTS-galactoside at concentrations ranging from 50 to 400 µM blocked reactivity completely. In contrast, at 50 µM MTS-glucoside, little or no effect on NEM labeling was observed, 100 µM blocked NEM labeling by about 50%, and complete abrogation was observed at 400 µM. Thus, there is good correlation between inhibition of transport activity and reactivity with the Cys residue at position 122. Consistent with this interpretation, under the same conditions, no inactivation whatever was observed with functional LacY devoid of Cys residues (data not shown).
If MTS-galactoside is an affinity reagent for the A122C/C148A mutant,
altering the affinity of LacY for substrate should be reflected in the
time course of MTS-galactoside inactivation. As shown in Fig.
3A and Table I, mutants
containing a single Cys residue at position 122 and Ala, Ser, or Asp at
position 148 exhibit decreasing initial rates of lactose transport.
Moreover, TDG protection against NEM labeling of Cys122
(24, 35) reveals Kd values of 0.2, 3, and 40 mM, respectively (Table I). Although there is no
quantitative relationship between the initial rate and
Kd, qualitatively, A122C/C148A LacY exhibited the
best affinity and the highest rate of transport followed by mutants
C148S and C148D (Fig. 3A; Table I). Relative to mutant
A122C/C148A, which exhibits a second-order rate constant (k)
of ~235 M1 s
1 for
MTS-galactoside inactivation, mutants A122C/C148S and A122C/C148D exhibit rate constants of about 56 and 31 M
1
s
1, respectively (Fig. 3B; Table I),
indicating that the reaction rates correlate qualitatively with binding
affinity and transport activity. In contrast, inactivation by
MTS-glucoside exhibits essentially the same rate of inactivation of
lactose transport by the three mutants and a second-order rate constant
that averages 11 M
1 s
1 (Fig.
3C; Table I). Thus, MTS-galactoside inactivates mutant A122C/C148A a minimum of 21 times faster than MTS-glucoside. Taken together, the results indicate that MTS-galactoside is an
affinity reagent. In contrast, MTS-glucoside reacts nonspecifically
like many other thiol-reactive reagents.
|
1
s
1 (Fig. 4A;
Table II). Remarkably, in the presence of
1 s
1 (Fig. 4A;
Table II). However, inactivation does not go to completion but plateaus
at 10 s because the concentration of the inactivator is depleted
due to reaction with Cys residues unassociated with LacY.2
In any case,
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The effect of
Inactivation by MTS-galactoside Occurs at a Step Subsequent to
Binding--
Presumably, MTS-galactoside or MTS-5-galactose reacts
directly upon binding. However, the marked increase in the rate of
reaction in the presence of -D-galactopyranoside (NPG) by TDG as demonstrated by
flow dialysis (Fig. 6A),
reflecting increased binding affinity. Although not shown, it is highly
noteworthy that specificity is maintained, as evidenced by the
observation that 10 mM glucose or sucrose has no effect. In
contrast, the mutant exhibits little or no active lactose transport
(Fig. 6B; Table I). In addition, E. coli HB101
(lacY
lacZ+) expressing
the mutant catalyzes almost no downhill translocation of the
disaccharide (data not shown).
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Because mutant A122C/C148A/C154G has essentially no transport activity,
reaction of MTS-galactoside with Cys122 was studied by
blockade of NEM labeling. Surprisingly, when RSO vesicles are incubated
with given concentrations of MTS-galactoside or MTS-glucoside, it is
apparent that the mutant essentially loses the ability to distinguish
between the MTS sugars; the EC50 is about 60 µM for both (Table I). At 200 µM, there is
no detectable difference between the rate of reaction of the mutant
with MTS-galactoside and MTS-glucoside (Fig. 6C).
Furthermore, similar results are obtained with MTS-5-galactose and
MTS-5-glucose (data not shown). Finally, as expected,
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DISCUSSION |
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Previous experiments (24) with A122C LacY demonstrate that the
galactopyranosyl moiety of disaccharide substrates of LacY is in close
proximity to Cys148 in helix V, whereas the non-galactosyl
moiety abuts Ala122. On the basis of these observations,
we synthesized the MTS sugars described here, demonstrating that
both MTS-galactoside and MTS-5-galactose inactivate mutant A122C at
least 20 times more rapidly than the corresponding glucose derivatives.
Furthermore, it is clear that inactivation of transport is due
specifically to reaction of the inactivators with the Cys residue in
place of Ala122. Thus, these results presented provide
strong confirmation of the previous conclusion (Fig.
7).
|
One noteworthy aspect of the findings is the difference in potency between the two sets of MTS sugars. Clearly, the ethylene linkage in MTS-galactoside and -glucoside confers greater hydrophobicity than the carbamide linkage in the MTS-5-galactose and -glucose derivatives. The preferential reaction of LacY with hydrophobic thiol reagents (46) is a likely explanation for the differences observed. It may also be relevant that the distance of the reactive thiol in the MTS-5 sugars is significantly further from the C-1 position of the galactopyranosyl ring (Fig. 1).
At first glance, MTS-galactoside and MTS-5-galactose appear to act as straightforward affinity reagents. Both compounds inactivate transport at concentrations at which the corresponding glucose derivatives have no effect. Moreover, when Cys148 is replaced with Ala, Ser, or Asp, there is a qualitative relationship between the respective affinities and the rates of transport and inactivation by MTS-galactoside. In contrast, transport in all three mutants is inactivated at the same rate by MTS-glucoside.
Remarkably, the rate of inactivation by MTS-galactoside and
MTS-5-galactose is increased by about 50- and 30-fold, respectively, in
the presence of a
Although it is possible that MTS-galactoside and MTS-5-galactose are
actively transported by LacY, it is unlikely that the marked
enhancement of inactivation rates by
It is also unlikely that
Mutation C154G results in a phenotype that binds ligand but does little
or no translocation of lactose across the membrane (42, 43, 44), and
the protein appears to be locked in an outwardly facing conformation
(44). When this mutation is combined with the A122C/C148A mutations,
LacY binds ligand with about 10 times greater affinity than mutant
A122C/C148A but catalyzes essentially no lactose translocation.
Furthermore, binding specificity is retained, because glucose or
sucrose at high concentrations does not displace bound NPG.
Surprisingly, however, as judged by NEM labeling (Fig. 4, C
and D), mutant A122C/C148A/C154G no longer discriminates
between MTS-galactoside and MTS-glucoside, and as expected, there is no
effect of
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ACKNOWLEDGEMENTS |
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We thank Yonglin Hu and Vladimir Kasho for the computer graphics shown in Fig. 7 and Gérard Leblanc for kindly providing [3H]NPG. We also thank Arthur Karlin for insisting on the calculation of rate constants.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK51131-07 and Grant OTKA T34307 from the Hungarian National Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: 5-748 Macdonald Research Laboratories, Box 951662, Los Angeles, CA 90095-1662. Tel.: 310-206-5053; Fax: 310-206-8623; E-mail: RonaldK@HHMI.UCLA.edu.
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M211355200
2 The concentration of N-ethylmaleimide-reactive Cys residues in right-side-out vesicles is approximately 20 nmol/mg protein (48). Therefore, the concentration of membrane-associated thiol groups per individual reaction mixture is about 60 µM.
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ABBREVIATIONS |
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The abbreviations used are:
LacY, lactose
permease;
MTS, methanethiosulfonate;
TDG, -D-galactopyranosyl
1-thio-
-D-galactopyranoside;
NPG, p-nitrophenyl
-D-galactopyranoside;
NEM, N-ethylmaleimide;
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42. | Menick, D. R., Sarkar, H. K., Poonian, M. S., and Kaback, H. R. (1985) Biochem. Biophys. Res. Commun. 132, 162-170[Medline] [Order article via Infotrieve] |
43. | van Iwaarden, P. R., Driessen, A. J., Lolkema, J. S., Kaback, H. R., and Konings, W. N. (1993) Biochemistry 32, 5419-5424[Medline] [Order article via Infotrieve] |
44. | Smirnova, I., and Kaback, H. R. (2003) Biochemistry, in press |
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47. | Robertson, D. E., Kaczorowski, G. J., Garcia, M. L., and Kaback, H. R. (1980) Biochemistry 19, 5692-5702[Medline] [Order article via Infotrieve] |
48. | Kaback, H. R., Reeves, J. P., Short, S. A., and Lombardi, F. J. (1974) Arch. Biochem. Biophys. 160, 215-222[Medline] [Order article via Infotrieve] |
49. |
Sorgen, P. L.,
Hu, Y.,
Guan, L.,
Kaback, H. R.,
and Girvin, M. E.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
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50. | Weinglass, A. B., Whitelegge, J. P., Hu, Y., Verner, G. E., Faull, K. F., and Kaback, H. R., (2003) EMBO J., in press |
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