Aromatic Stacking in the Sugar Binding Site of the Lactose Permease†
Lan Guan, Yonglin Hu, and H. Ronald Kaback*
Howard Hughes Medical Institute, Departments of Physiology and Microbiology and Molecular Genetics,
Molecular Biology Institute, UniVersity of California Los Angeles, Los Angeles, California 90095-1662
ReceiVed NoVember 11, 2002; ReVised Manuscript ReceiVed December 16, 2002
ABSTRACT: Major determinants for substrate recognition by the lactose permease of Escherichia coli are
at the interface between helices IV (Glu126, Ala122), V (Arg144, Cys148), and VIII (Glu269). We
demonstrate here that Trp151, one turn of helix V removed from Cys148, also plays an important role in
substrate binding probably by aromatic stacking with the galactopyranosyl ring. Mutants with Phe or Tyr
in place of Trp151 catalyze active lactose transport with time courses nearly the same as wild type. In
addition, apparent Kmvalues for lactose transport in the Phe or Tyr mutants are only 6- or 3-fold higher
than wild type, respectively, with a comparable Vmax. Surprisingly, however, binding of high-affinity
galactoside analogues is severely compromised in the mutants; the affinity of mutant Trp151fPhe or
Trp151fTyr is diminished by factors of at least 50 or 20, respectively. The results demonstrate that
Trp151 is an important component of the binding site, probably orienting the galactopyranosyl ring so
that important H-bond interactions with side chains in helices IV, V, and VIII can be realized. The results
are discussed in the context of a current model for the binding site.
Like many membrane transport proteins comprising the
Major Facilitator Superfamily (MFS)1(1), the lactose per-
mease of Escherichia coli (LacY) transduces free energy
stored in an electrochemical H+gradient into a concentration
gradient of D-galactopyranosides and vice versa (galactoside/
H+symport) (2). LacY is a 12-transmembrane-helix bundle
with the N and C termini on the cytoplasmic face of the
membrane (3-5), and it is physiologically (6) and structur-
ally a monomer in the membrane (7-9). Analysis of an
extensive library of mutants, Cys-replacement mutants in
particular (10), with various site-directed biophysical and
biochemical techniques has led to the formulation of a tertiary
structure model (11), as well as a hypothesis for the
mechanism of lactose/H+symport (12).
LacY is selective for disaccharides containing a D-
galactopyranosyl ring, as well as D-galactose (13-15), but
does not interact with D-glucopyranosides or D-glucose (15-
17). The specificity of LacY is directed toward the galac-
topyranosyl ring of the substrate, and although C-4 OH is
by far most important for specificity, the C-2, C-3, and C-6
OH groups are also important for binding (C-4 OH . C-3
OH g C-6 OH > C-2 OH) (15, 17).
The major determinants for the substrate binding site are
located at the interface between helices IV, V, and VIII.
Glu126 (helix IV) and Arg144 (helix V), which are charge-
paired, are irreplaceable with respect to substrate binding; a
carboxyl group at position 126 and a guanidino group at
position 144 are absolute requirements, and the side chains
are located at the interface between helices IV and V,
respectively (Figure 1) (18-22). Although Cys148 (helix V)
is not irreplaceable, it interacts weakly and hydrophobically
with the galactopyranosyl moiety of LacY substrates (16,
23-26), and Ala122 (helix IV), another nonessential residue,
abuts the nongalactopyranosyl moiety of the disaccharide
substrates (22, 27, 28). Thus, alkylation of mutant A122C
or replacement with Phe or Tyr abrogates disaccharide
binding and transforms LacY into a galactose-specific
symporter (22). This conclusion receives strong support from
recent experiments (28) demonstrating that 1-methanethio-
sulfonyl-?-D-galactopyranosyl derivatives are affinity in-
activators for mutant A122C. Finally, recent experiments
utilizing carbodiimide labeling in the absence and presence
of ligand in conjunction with mass spectroscopy indicate that
Glu269 (helix VIII) interacts directly with the galactopyra-
nosyl ring (Weinglass, A. B., Whitelegge, J. P., Hu, Y., Faull,
K. F., and Kaback, H. R., submitted for pubication).
Although it has been suggested (see ref 22) that Cys148
interacts with the hydrophobic face of the galactopyranosyl
ring, in this configuration the C-4 OH (which is the most
important OH group in the ring) cannot interact with Arg144,
its postulated H-bond partner, nor can the C-6 OH interact
with Glu126 (see ref 19). However, by positioning the
†This work was supported in part by NIH Grant DK51131:07 to
* To whom correspondence should be addressed. Telephone:
(310) 206-5053. Telefax:(310) 206-8623. E-mail:
1Abbreviations: MFS, Major Facilitator Superfamily; LacY, lactose
permease; TDG, ?-D-galactopyranosyl 1-thio-?-D-galactopyranoside;
NPG, p-nitrophenyl R-D-galactopyranoside; NEM, N-ethylmaleimide;
KPi, potassium phosphate.
Biochemistry 2003, 42, 1377-1382
10.1021/bi027152m CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/23/2003
galactopyranosyl ring at a right angle with helix V so that
Cys148 is close to the 1 position (Figure 1), many of the
postulated interactions are fulfilled. Furthermore, when the
binding site is modeled in this fashion, it becomes clear that
Trp151 is in excellent position to stack hydrophobically with
the galactopyranosyl moiety of LacY substrates, a common
feature of carbohydrate binding sites in over 50 proteins of
known structure (see refs 29 and 30-34), as well as
maltoporin (35, 36) where aromatic side chains play an
important role in the translocation pathway.
On the other hand, previous studies (37) in which each
Trp residue in LacY was replaced with Phe suggest that none
of the Trp residues is essential for activity. Moreover, it was
demonstrated that mutant W151F seemingly catalyzes active
lactose transport as well as the wild type at a lactose
concentration approximating the Km, thereby suggesting that
further studies such as kinetics or ligand binding would be
unrevealing. In view of the considerations discussed above,
however, studies on Trp151 were reinitiated. Remarkably,
despite excellent transport activity in mutants W151F and
W151Y with relatively small alterations in kinetic param-
eters, both mutants exhibit markedly decreased substrate
affinity, and mutant W151I exhibits an affinity that is too
low to measure accurately. The results support a model for
the binding site in which Trp151 plays an important role in
ligand binding, interacting hydrophobically with the galac-
topyranosyl ring and holding it in an orientation that allows
H-bond interactions required for specificity.
Materials. [1-14C]lactose was obtained from Amersham
Pharmacia Biotech (Piscataway, NJ). N-([14C]ethyl)male-
imide was purchased from Dupont NEN (Boston, MA).
p-Nitrophenyl R-D-[6-3H]galactopyranoside (NPG) was the
generous gift of Ge ´rard Leblanc (Villefranche-sur-mer,
France). Immobilized monomeric avidin was from Pierce
(Rockford, IL), and all unlabeled sugars were obtained from
Sigma (St. Louis, MO). Oligodeoxynucleotides were syn-
thesized by Sigma-Geneosys (The Woodlands, TX). Restric-
tion endonucleases, T4 DNA ligase, and Vent DNA poly-
merase were from New England Biolabs (Beverly, MA). All
other materials were reagent grade and obtained from
FIGURE 1: Proposed binding site in LacY showing helices IV, V, and VIII with bound lactose. The galactopyranosyl ring, which contains
all the determinants for specificity, is shown in a ?-1,4 linkage with D-glucose. The C-4 OH is most important with respect to specificity
(17). Cys148 (helix V) interacts weakly and hydrophobically with the galactopyranosyl ring, and Ala122 is in close proximity to the
glucopyranosyl moiety (22). Data presented here indicate that Trp151 stacks with the hydrophobic face of the galactopyranosyl ring, placing
it at a right angle with helix IV and abutting Cys148 near the 1 position. In this orientation, the C-4 OH can H-bond directly with either
NH1or NH2of Arg144, and the C-6 OH can H-bond with Glu126. Since the C-3 OH is close to Glu269 (helix VIII) but at an angle, it is
a reasonable suggestion that a water molecule may mediate this interaction.
1378 Biochemistry, Vol. 42, No. 6, 2003
Construction of LacY Mutants. Oligonucleotide-mediated,
site-directed mutagenesis by using two-step PCR with pT7-
5/cassette lacY as template was applied to construct Tyr, Phe,
and Ile replacements for Trp151. In addition, pKR35
plasmids encoding LacY mutants W151Y/single-Cys148,
W151F/single-Cys148, and W151I/single-Cys148 mutants
were generated in a similar manner by using pKR35 encoding
single-Cys148 with a C-terminal biotin acceptor domain as
template (22, 38-40).
Growth of Cells. E. coli T184 [lacI+O+Z-Y-(A)
taining given mutants was grown in Luria-Bertani broth with
100 mg/L 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
Preparation of Right-Side-Out (RSO) Membrane Vesicles.
RSO membrane vesicles were prepared by osmotic lysis as
described (41, 42), suspended in 100 mM potassium phos-
phate KPi(pH 7.5)/10 mM MgSO4at a protein concentration
of about 12 mg/mL, frozen in liquid N2, and stored at -80
°C until use.
Transport Assays. E. coli T184 containing given LacY
mutants were washed with 100 mM KPi(pH 7.5)/10 mM
MgSO4and adjusted to an OD420of 10.0 (0.7 mg protein/
mL). Transport was carried out with [1-14C]lactose (10 mCi/
mmol) at a final concentration of 0.4 mM. Lactose transport
in RSO membrane vesicles was assayed in the presence of
20 mM ascorbate/0.2 mM phenazine methosulfate under
oxygen with given concentrations of [1-14C]lactose (10 mCi/
Binding Affinity Measurements. The KDfor TDG binding
was determined in situ by alkylation of given mutants with
0.5 mM [14C]NEM (40 mCi/mmol) in the absence or
presence of given concentrations of ?-D-galactopyranosyl
1-thio-?-D-galactopyranoside (TDG) (15, 17, 25, 44-46).
Flow Dialysis. Binding of [3H]NPG to RSO vesicles
containing given LacY mutants was measured by flow
dialysis as described (47).
ActiVe Transport. As found previously (37), E. coli T184
(lacZ-Y-) expressing W151Y or W151F LacY seemingly
catalyzes lactose transport at a rate and to a steady-state level
of accumulation comparable to cells expressing wild-type
LacY (Figure 2). However, when initial rates are measured
carefully over the first 15 s, an approximate 2-fold decrease
is observed with mutants W151Y and W151F (Figure 2,
inset; Table 1).
Kinetic Analyses of Lactose Transport. Initial rates of
lactose transport measured over the first 15 s increase in
hyperbolic fashion as a function of lactose concentration in
wild-type LacY, as well as mutants W151F and W151Y
(Figure 3A). Furthermore, as shown in Figure 3B, linear
functions are obtained in Eadie-Hofstee plots. The apparent
Kmfor wild-type LacY derived from the slope is 0.43 mM,
and the Vmax estimated from the y intercept is 250 nmol/
min/mg protein. The apparent Km for mutant W151F or
W151Y is 2.45 or 1.22 mM, respectively, which is only 6-
or 3-fold higher than wild-type LacY (Table 1). The transport
activity of mutant W151I is too low for accurate kinetic
studies (data not shown).
NPG Binding. As shown by flow dialysis measurements
with RSO membrane vesicles using [3H]NPG (Figure 4),
wild-type LacY exhibits a relatively large increase in the
dialysable concentration of [3H]NPG after addition of a
saturating concentration of TDG, indicating good binding
affinity for NPG. In contrast, with either mutant W151F or
W151Y, only a small increase in the dialyzable NPG
concentration is observed upon addition of TDG, indicating
that NPG binding is largely compromised.
Substrate Protection Against Alkylation of Cys148 by
NEM. To determine disassociation constants (KD), the
Typ151 replacement mutations were placed in the single-
Cys148 background, and TDG-dependent protection of
Cys148 against alkylation by [14C]NEM was quantified. The
pseudo-wild type (single-Cys148 LacY with Trp151) exhibits
a KDof 35 µM (Figure 5A; Table 1). Remarkably, mutant
W151F (Figure 5B; Table 1) or W151Y (Figure 5C; Table
1) exhibits KDvalues of about 1.8 or 0.7 mM, respectively,
which corresponds to decreases in affinity by factors of about
50 or 20 relative to the pseudo-wild type (Table 1). Although
data are not presented, the W151I mutant exhibits a KDwell
in excess of 30 mM.
As shown in the earlier studies (37), the overall time course
of lactose transport by mutants W151F and W151Y at a
transport. E. coli T184 expressing wild-type permease or given
mutants at position 151 were grown at 37 °C, and an aliquot of
cell suspension (50 µL containing approximately 35 µg of protein)
in 100 mM KPi(pH 7.5)/10 mM MgSO4was assayed at 0.4 mM
final lactose concentration as described in Experimental Procedures.
Inset: initial rates of lactose transport measured over the first 15
s. 9, wild type; b, W151Y; 2, W151F; and 1, pT7-5 with no
Effect of Trp151 replacements on active lactose
Table 1: Effect of Trp151 Mutations on Kinetics and Binding
aLactose transport and Kmvalues were measured in given mutants
as described in Figures 2 and 3.bKDvalues were measured in single-
Cys148 background as described in Figure 5.cValue for single-Cys148
Biochemistry, Vol. 42, No. 6, 2003 1379
lactose concentration that approximates the Kmis similar to
that of the wild type (40, 45). However, careful measure-
ments of initial rates display a modest decrease in the
mutants. Consistently, kinetic analyses reveal relatively minor
increases in Km, only about a 6-fold increase for mutant
W151F and a 3-fold increase for mutant W151Y (Figure 3;
Table 1). Although there is a relatively small difference in
the kinetics of transport in the W151F and W151Y mutants,
it is apparent that the presence of a Trp at position 151 gives
the wild type a distinct advantage in the natural environment.
Strikingly and unexpectedly, however, both mutants
exhibit dramatic decreases in binding affinity, at least a 50-
FIGURE 3: Effect of Trp151 replacements on kinetics of lactose
transport. RSO membrane vesicles prepared from E. coli T184
containing given LacY mutants were assayed for lactose transport
as described in Experimental Procedures. Initial rates (V) were
measured at 0, 5, 10, and 15 s and plotted as function of V/S, where
S represents lactose concentration. Inset: initial rates plotted directly
as a function of lactose concentrations. 9, wild type; 2, W151F;
and b, mutant W151Y.
FIGURE 4: Effect of Trp151 mutations on NPG binding. Binding
of [3H]NPG to nonenergized RSO vesicles with wild-type permease
(9), W151F (2), or mutant W151Y (b) at a protein concentration
of 32 mg/mL was assayed by flow dialysis. [3H]NPG (840 mCi/
mmol) at 15 µM final concentration was added at fraction 1. As
indicated by the arrow, TDG at 10 mM final concentration was
added at fraction 9 to displace bound NPG.
FIGURE 5: Substrate protection against [14C]NEM labeling of single-
Cys148 LacY with given replacements for Trp151. RSO membrane
vesicles containing given LacY mutants incubated with 0.5 mM
[14C]NEM (40 mCi/mmol) for 5 min at pH 7.5 in absence or
presence of the given concentrations of TDG. Reactions were
quenched with 10 mM DTT, and biotinylated permease was
solubilized in dodecyl ?,D-maltopyranoside and purified by affinity
chromatography on monomeric avidin Sepharose. Samples were
subjected to sodium dodecyl sulfate/12% polyacrylamide gel
electrophoresis, and [14C]labeled protein was quantitated with a
Storm 860 PhosphoImager. Labeling in the presence of a given
concentration of sugar is expressed as percent labeling observed
in the absence of the sugar. KDvalues were determined with the
ORIGIN computer program (Microcal Software, Northampton, MA)
by using nonlinear least-squares curve fitting to the following user-
defined equation: Y ) (1 - P1)/(1 + X/P2) + P1, where P1 is the
residual labeling and P2 is the KD.
1380 Biochemistry, Vol. 42, No. 6, 2003
fold increase in KD for TDG in mutant W151F and a 20-
fold increase in W151Y (Table 1). It is noteworthy that the
order of efficacy of the side chain at position 151 (Trp .
Tyr > Phe) is consistent with the aromaticity of the three
residues. Thus, the distance and orientation of Trp151 is
optimal for van der Waals interactions followed by Tyr and
Phe. The results clearly support the notion that Trp151 stacks
hydrophobically with the galactopyranosyl ring (Figure 1).
By this means, Trp151 orients the galactosyl ring in such a
position that important H-bond interactions between the OH
groups on the ring and the side chains on LacY can be
formed. In this regard, recent studies (Weinglass, A. B.,
Whitelegge, J. P., Hu, Y., Faull, K. F., and Kaback, H. R.,
submitted for pubication) demonstrating ligand protection
of Glu269 (helix VIII) against modification by hydrophobic
carbodiimides by mass spectrometry also suggest that this
residue may form an H-bond with the C-3 OH of the
galactopyranosyl ring. Moreover, since the C-3 OH is close
to Glu269 but at an angle (Figure 1), it is reasonable to
suggest that a water molecule may mediate the interaction.
For additional clarity, a side view of the putative binding
site depicting only helices IV and V is presented in Figure
6 with the sugar shown in space-filling form and relevant
side chains as balls and sticks. In addition to illustrating
clearly how the hydrophobic face of the galactopyranosyl
ring might stack hydrophobically with Trp151, there appears
to be a hydrophobic notch with the aromatic side chain
forming a shelf and the side of the galactopyranosyl ring
interacting with Cys148 in the vicinity of the 1 position of
Since Trp residues tend to localize at the membrane/
aqueous interface in known 3-D structures of membrane
proteins (48), it is of interest that out of the six Trp residues
in LacY, five are located at or near the ends of helices, while
Trp151 is present within transmembrane helix V (49, 50).
Furthermore, multiple sequence alignment of the 10 members
of the Oligosaccharide/H+Symport subfamily of the MFS
(Clustal W) reveals that the amino acid side chains involved
in substrate specificity are conserved to a high degree in all
orthologues of LacY that recognize galactopyranosides. Thus,
a Glu residue is present at position Glu126, and an Arg
residue is conserved at position 144 in all instances. A Cys
residue is largely conserved at position 148, an aromatic
groups Trp or in two instances Tyrsis present at position
151, and Glu269 is also very largely conserved. In contrast,
Ala122, which interacts with the nongalactopyranosyl moiety
of substrate and plays little or no role in specificity, is found
in only three homologues, with Ser in four and Ala or Cys
in two others. It is also noteworthy that the remaining
irreplaceable residuessArg302, His322, and Glu325swhich
are involved in H+translocation but do not make direct
contact with the galactopyranosyl moiety of substrate are
also highly conserved. Therefore, as in the case of soluble
sugar binding proteins, it seems likely that aromatic stacking
may play an important role in sugar binding by membrane
transport proteins as well.
We thank Ge ´rard Leblanc for generously providing [3H]-
NPG and Melissa Sondej for providing RSO vesicles
containing wild-type LacY.
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