From the Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030
Received for publication, December 26, 2000, and in revised form, January 8, 2001
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ABSTRACT |
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In Escherichia coli, interaction of a
periplasmic maltose-binding protein with a membrane-associated
ATP-binding cassette transporter stimulates ATP hydrolysis, resulting
in translocation of maltose into the cell. The maltose transporter
contains two transmembrane subunits, MalF and MalG, and two copies of a
nucleotide-hydrolyzing subunit, MalK. Mutant transport complexes that
function in the absence of binding protein are thought to be stabilized
in an ATPase-active conformation. To probe the conformation of the
nucleotide-binding site and to gain an understanding of the nature of
the conformational changes that lead to activation, cysteine 40 within
the Walker A motif of the MalK subunit was modified by the fluorophore
2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid. Fluorescence
differences indicated that residues involved in nucleotide binding were
less accessible to aqueous solvent in the binding protein independent
transporter than in the wild-type transporter. Similar differences in
fluorescence were seen when a vanadate-trapped transition state
conformation was compared with the ground state in the wild-type
transporter. Our results and recent crystal structures are consistent
with a model in which activation of ATPase activity is associated with
conformational changes that bring the two MalK subunits closer
together, completing the nucleotide-binding sites and burying ATP in
the interface.
The periplasmic binding protein-dependent maltose
transport system of Escherichia coli is a member of the
ATP-binding cassette or ABC1
superfamily of proteins. Members of this protein family are responsible for the transport of a variety of substrates across membranes in both
prokaryotic and eukaryotic organisms (1, 2). Medically important
members of the superfamily include the multidrug-resistant P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (3, 4). A soluble maltose-binding protein (MBP) and a
membrane-spanning transport complex consisting of one MalF, one MalG,
and two MalK (MalFGK2) protein subunits mediate maltose transport across the cytoplasmic membrane. The hydrophobic MalF and
MalG proteins are predicted to form a transmembrane channel for
substrate, and ATP hydrolysis by the associated MalK subunits is
coupled to the translocation event (5, 6).
Periplasmic binding proteins bind their substrates tightly and are
responsible for the high affinity of this class of prokaryotic transporters (7). In the presence of ligand, the binding proteins undergo a conformational change (8, 9) and initiate translocation by
stimulating the ATPase activity of the membrane-bound transporters (10). We have recently demonstrated that MBP activates the ATPase activity of MalFGK2 by stabilizing the transition state for
ATP hydrolysis, and as a consequence becomes tightly bound to the transport complex in the presence of the transition state analogue vanadate (11). Vanadate traps ADP in one of the two nucleotide-binding sites, locking the transport complex in the transition state
conformation (11, 12). Maltose is likely passed from MBP to
MalFGK2 in the transition state, thereby coupling transport
to ATP hydrolysis. Mutations have been isolated in MalF and MalG that
permit maltose transport in the absence of MBP, albeit with lowered
affinity (7). These binding protein-independent (BPI) transport
complexes have gained the ability to hydrolyze ATP in the absence of
MBP (14). We propose that the BPI mutants function by stabilizing the
transport complex in a conformation that is associated with the
activation of the ATPase activity of the WT. If the WT and BPI
complexes represent the ground state and an active state of the
transport complex, respectively, detection of conformational differences between the two complex types could provide information about the transport mechanism.
The fluorescence of the sulfhydryl-specific reagent MIANS is sensitive
to the polarity of its environment (15, 16) and has been used to detect
conformational changes in the Na+,K+-ATPase
(15), the myosin ATPase (17), the P-glycoprotein (18, 19), and the
lactose permease (20, 21). In this study, we used MIANS to detect
conformational differences in the nucleotide-binding sites of WT and
BPI complexes in the ground state and in the transition state to gain
further insight into the mechanism of activation of MalFGK2
ATPase activity. We found that the conformation of the
nucleotide-binding sites in the BPI complex more closely resembles the
transition state than the ground state of the WT complex. Furthermore,
the conformational change associated with activation of the ATPase
activity relocates residues involved in nucleotide binding to a less
surface-exposed environment.
Bacterial Strains, Plasmids, and Culture Conditions--
Strain
HN741 (Escherichia coli K-12 argH his rpsL1 malT
(Con) malB Protein Purification--
Histidine-tagged transport complexes
were purified by affinity chromatography as published previously (23)
with modifications. Briefly, thawed membrane vesicles were washed by
dilution in 20 mM HEPES (pH 8), 1 mM EDTA and
collected by centrifugation at 100,000 × g for 30 min.
Vesicles were resuspended at a protein concentration of ~3 mg/ml in
50 mM NaPi (pH 8), 10% glycerol, and membrane
proteins were solubilized by the addition of n-dodecyl MIANS Modification and ATP Protection
Studies--
2-(4'-Maleimidylanilino)naphthalene-6-sulfonic acid
(MIANS) was purchased from Molecular Probes, Inc. (Eugene, OR). For
MIANS modification studies, transport complexes were labeled in
detergent solution (buffer B) by incubation at 25 °C with either 5 or 20 µM MIANS added from a 0.2 mM stock
solution in methanol. Complexes that had been reconstituted into
proteoliposome vesicles by dilution (6) were labeled in buffer
containing 20 mM NaPi (pH 8) and 10 mM MgCl2 at 37 °C (26). In control
incubations methanol was added to complexes to the same percent volume
as the MIANS stock solution. For ATP protection experiments, complexes
were briefly preincubated with ATP as specified in the figure legends
before the MIANS was added. Following incubation for 15 min at the
specified temperature, reactions were terminated with 1 mM
DTT, and complexes were assayed for ATPase activity.
Assays of ATP Hydrolysis--
ATP hydrolysis activity of
detergent-soluble and reconstituted transport complexes was measured
using [ Fluorescence Measurements--
For fluorescence studies,
transport complexes were incubated with MIANS for 15 min at 4 °C.
Excess MIANS was removed from the protein sample using a HiTrap
desalting column (Amersham Pharmacia Biotech). Fluorescence
measurements were made using an ISS PC1 Photon Counting
Spectrofluorometer (Champaign, IL). Fluorescence spectra for MIANS
reacted with Preparation of Vanadate-trapped Complexes for Solvent
Accessibility Studies--
WT and BPI complexes were treated with
vanadate in detergent solution as described previously (11) with
modifications. Briefly, reactions contained either 2.5 µM
WT or BPI transport complex in buffer B containing 4 mM
ATP; 5 µM MBP and 0.01 mM maltose were also
included in the WT reactions as specified in the figure legends.
Vanadate was added to a final concentration of 0.5 mM where
indicated, and the reactions were incubated for 20 min at 37 °C. The
samples were then desalted using a Hitrap column and labeled with MIANS
as described above. Stable association of MBP with the trapped WT
complexes was confirmed using ion-exchange chromatography and SDS-PAGE
(11).
Electrophoresis--
SDS-polyacrylamide gel electrophoresis was
performed with 11% polyacrylamide gels using the procedure described
by Laemmli (29). Fluorescent bands on gels were visualized in 5%
methanol, 7.5% acetic acid (17) over an ultraviolet light source and
recorded using a Nucleovision imaging system.
Protein Determinations--
Protein concentrations were
determined as described previously (6) using the method of Schaffner
and Weissmann (30).
Specific Modification of MalK Residue Cys-40 by MIANS--
In an
effort to detect conformational changes associated with activation of
the ATPase activity of MalFGK2, we wished to modify specifically a cysteine residue in the MalK subunit(s) with a fluorescent reporter group. The MalFGK2 complex contains a
total of 10 cysteine residues (three in each MalK, three in MalF, and one in MalG (31-33)), including a reactive cysteine close to the nucleotide-binding site. This residue is located at position 40 (Cys-40) within the Walker A consensus nucleotide-binding motif (also
known as the phosphate binding loop, or P-loop) of MalK (34).
Modification of this residue by the sulfhydryl-specific reagent
N-ethylmaleimide (NEM) inhibits the ATPase activity of the
maltose transport complex (35, 36); ATP protects against inhibition
(35), and replacement of this cysteine with serine eliminates
inhibition without affecting normal activity (37). Based on these
findings, we predicted that it would be possible to label covalently
Cys-40 with MIANS, a fluorescent maleimide probe that could be used to
monitor conformational changes in the MalK subunit. Like NEM, MIANS
also inhibited the ATPase activity of both WT and BPI transport systems
(Table I). MIANS did not inhibit the
ATPase activity of complexes containing a nondeleterious substitution
of cysteine with glycine at position 40 in MalK (C40G) (24). These
results demonstrate that MIANS reacts with Cys-40 and that modification
at this site is responsible for the inhibition of ATPase activity.
To interpret possible conformational differences detected by MIANS as
being associated with Cys-40 in the nucleotide-binding site(s), we
assessed the extent of labeling of the other cysteines in the transport
complex both by SDS-PAGE and by comparison of the intensity of the
fluorescence emission spectra of complexes containing either Cys-40 or
a C40G substitution. As seen in SDS-PAGE analysis of MIANS-labeled
complexes, very little fluorescence is associated with the MalF and
MalG proteins, and substitution of a glycine for the cysteine at
position 40 greatly reduces the fluorescence associated with MalK (Fig.
1). The fluorescence emission spectra of
the MIANS-labeled WT and BPI complexes revealed that the fluorescence
of MIANS associated with C40G complexes was only 10-35% that seen for
Cys-40 complexes (Fig. 2). Thus, although residues other than Cys-40 may be modified to some extent, the bulk of
the fluorescence is associated with MIANS bound at Cys-40. Variation of
buffer, pH, MIANS concentration, and length of incubation did not
improve the specificity of the modification reaction (data not shown).
Quantitation of MIANS incorporation into MalFGK2 through absorbance measurements at 322 nm (38) failed to address the question
of whether residue Cys-40 was modified in one or both MalK subunits
(data not shown), in part because of the variability in the background
(non-Cys-40) labeling by MIANS. However, based on other observations
(see "Discussion"), we hypothesize that only one of the two MalK
subunits is modified.
Emission Maximum of BPI Is Blue-shifted Relative to WT--
The
fluorescence characteristics of MIANS are sensitive to the
hydrophobicity of its environment; specifically, emission maxima are
blue-shifted to shorter wavelengths, and the quantum yield increased as
the solvent polarity decreases (16, 17). The emission spectra in Fig. 2
were therefore used to compare the local environment of MIANS at Cys-40
in the WT and BPI complexes. For both complexes, MIANS exhibited a
greater fluorescence quantum yield when bound to protein than when
reacted with Solvent Accessibility of Cys-40 in BPI Is Reduced Compared with
WT--
To probe further the nature of the conformational difference
suggested by the blue shift in the fluorescence emission of the BPI
transport complex, collisional quenching experiments were performed to
assess the solvent accessibility of MIANS. Specifically, the ability of
water-soluble reagents to collide with MIANS and quench its
fluorescence was measured as a function of quencher concentration to
compare the relative exposure of MIANS in WT and BPI complexes to the
aqueous environment. The solvent accessibility of MIANS complexes was
assessed using either acrylamide (Fig. 3,
A and B) or DPX (Fig. 3, C and
D) as a quenching agent. As anticipated, MIANS bound to the
complexes was less accessible to the quenching reagents than MIANS free
in solution (KSV = 3.18 M
In experiments similar to those presented here, the fluorescence of
MIANS positioned in the P-loop of P-glycoprotein was quenched by ATP in
a concentration-dependent manner (18). We therefore added
ATP to the labeled MalFGK2 complexes to determine whether nucleotide binding could be detected as a change in the fluorescence properties of MIANS at Cys-40. The presence of 1 mM ATP had
no effect on the fluorescence emission spectrum of either transport complex (data not shown) and did not significantly alter solvent accessibility (Fig. 3, B and D) other than a
small effect on exposure of the WT to acrylamide. Because ATP protects
against modification of residue Cys-40 (35), MIANS might prevent
nucleotide binding in the MalFGK2 system via a steric
effect. If MIANS modifies only one nucleotide-binding site, it is also
possible that ATP binds to one site without significantly altering the
fluorescence characteristics of MIANS at the second site.
Detection of Conformational Changes Associated with Attainment of
the Transition State for ATP Hydrolysis--
We recently showed that
vanadate inhibits MalFGK2 by trapping the complex in its
transition state conformation with ADP tightly bound in one of the two
nucleotide-binding sites (11, 12). Vanadate-induced nucleotide trapping
requires MBP in the WT but not in a BPI mutant (12). The ability to
trap the transition state provides a second model system that can be
used to study conformational changes associated with activation of
ATPase activity. Because vanadate trapping of the transition state
conformation requires ATP hydrolysis (12), complexes were first treated
with vanadate and then labeled with MIANS. The fluorescence intensity of either the WT or the BPI complex labeled after vanadate treatment was very similar to the intensity of control preparations labeled without the treatment; however, in the WT complex, vanadate treatment resulted in a 3-nm blue shift in fluorescence emission relative to the
untreated complex (data not shown). The solvent accessibility of MIANS
was then assessed using acrylamide quenching. The accessibility of
MIANS in the BPI complex was unchanged by vanadate treatment (Fig.
4). In the WT complexes, addition of MBP
in the absence of vanadate did not alter the solvent accessibility of
MIANS (Fig. 4). However, the solvent accessibility of MIANS was
different when WT complexes were vanadate-trapped in the presence of
MBP. The accessibility was reduced to a level resembling that of MIANS bound to BPI complexes, suggesting that the conformation of the BPI may
resemble the transition state of the WT transporter.
Another feature of the WT complex trapped in the transition state
species is that it binds tightly to MBP (11), whereas in the ground
state it displays a relatively low affinity for MBP (39, 40). If the
conformation of the BPI transporter resembles the transition state, it
might display a high affinity for MBP even in the absence of vanadate,
as shown indirectly for another BPI mutant, MalFG511K2
(40). As shown in Fig. 5, MBP is tightly associated with the MalF500GK2 BPI complex, both in the
presence and absence of treatment with vanadate, whereas the tight
binding interaction is seen for the WT only in the presence of
vanadate. This result provides additional evidence that the
conformation of the BPI complex resembles the transition state and may
in fact represent an intermediate in the pathway from the ground state to the transition state in the WT. As such, further comparison of WT
and BPI complexes might provide increased insight into the mechanism of
activation of MalFGK2 ATPase activity.
WT and BPI Complexes Differ in Affinity for ATP--
In
preliminary experiments, we noted a difference in the ability of ATP to
protect against the irreversible modification of the WT and BPI
complexes by MIANS (data not shown). To pursue this observation
further, the concentration dependence of ATP protection of WT and BPI
complexes against modification by either 20 or 5 µM MIANS
was examined (Fig. 6, A and
B). The concentrations of ATP needed to protect 50% of the
complexes against modification, or PC50 values, are
summarized in Table II. At the 20 µM MIANS concentration, 30-fold more ATP is required to
protect the WT transporters as opposed to the BPI transporters. The
same results are obtained at the 5 µM MIANS
concentration, although a correspondingly lower concentration of ATP is
required to gain the same level of protection. These results suggest
that MIANS and ATP compete for binding to MalFGK2 and that
the BPI mutant may have a higher affinity for ATP than the WT
transporter. In the absence of a reliable assay to measure nucleotide
binding to MalFGK2, we estimated the affinity indirectly by
measuring the half-saturation constant (K0.5)
for ATP hydrolysis by the BPI and WT complexes (Table II). The
K0.5 for the BPI transporter was 14-fold lower
than the corresponding value for the WT transporter and correlated well
with the ability of ATP to protect against MIANS modification. At the
lower concentration of MIANS (5 µM) the
PC50 values approached the K0.5,
suggesting that the differential ability of ATP to protect truly
reflects the differential nucleotide binding affinity of the WT and BPI complexes. These data suggest that BPI transporters have a greater affinity for ATP than do WT transporters.
We have used an extrinsic fluorescent probe to detect
conformational changes in the nucleotide-binding site associated with activation of the MalFGK2 ATPase. In the WT system, the
ATPase activity of the MalFGK2 transporter is stimulated
through an interaction with maltose-bound MBP that stabilizes the
transition state for hydrolysis (11). The need for MBP can be bypassed
by mutations (BPI mutations) that result in constitutive activation of
the ATPase activity of the transporter (14). A conformational
difference between the WT and BPI transport complex, detected in the
absence of both MBP and nucleotide, manifested itself both as a change in the position of the emission spectrum maximum and in the solvent accessibility of bound MIANS. The fluorescence of MIANS bound to
residue Cys-40 of MalK was blue-shifted in the BPI complexes relative
to the WT complexes, indicative of a more hydrophobic environment, and
was less accessible to water-soluble quenching agents. These results
suggest that residue Cys-40 in the P-loop is shifted to a more buried
location in the conformation that is stabilized by the BPI mutations.
In the case of the BPI MalF500GK2 complex used in this
study, two mutations in the transmembrane region of MalF, G338R and
N505I (41), are responsible for the conformational changes in the
nucleotide-binding site of the MalK subunit(s) and the activation of
the ATPase activity (14).
Because vanadate can be used to lock both the WT and the BPI
transporter in the transition state for ATP hydrolysis (11, 12), we
also used differences in the fluorescence characteristics of MIANS to
compare the ground state and transition state conformations of
MalFGK2. When vanadate was used to lock the WT
MalFGK2 complex in the transition state, the accessibility
of residue Cys-40 in the P-loop was decreased relative to the ground
state. This decreased level of accessibility was very similar to the
level observed for the ground state of the BPI complex, suggesting that
the conformational changes that activate the ATPase activity, whether
by interaction with MBP or by mutation, may be similar. In contrast,
vanadate trapping of the transition state of the BPI complexes had no
effect on the solvent accessibility of MIANS (Fig. 4), suggesting that the environment of residue Cys-40 does not change substantially in the
transition from the ground state to the transition state of the BPI
transporter. The absence of any effect in the BPI system argues that
steric effects of nucleotide trapped by vanadate in one of the two
nucleotide-binding sites (11) are not solely responsible for the
decrease in solvent accessibility of MIANS in the WT. Since ATP
protects against MIANS modification (Fig. 6), we predict that only the
unoccupied nucleotide-binding site is modified after vanadate
treatment. Since the fluorescence intensity of transporters was the
same whether they were labeled before or after vanadate-induced
nucleotide trapping, MIANS may modify only one nucleotide-binding site
even in the untreated transporter. Modification of a single
nucleotide-binding site by 4chloro-7-nitrobenzofurazan-Cl has
been demonstrated for P-glycoprotein, where it appears that covalent
modification of one site prevents modification of the other site
(42).
The similarities in the environment of the nucleotide-binding site in
the BPI mutant and in the transition state suggest that the mutations
in the BPI transporter have stabilized a conformation that resembles,
at least superficially, the conformation of the transition state,
thereby overcoming the major requirement for MBP in stabilization of
the transition state and therefore activation of the ATPase activity.
The observation that the BPI MalF500GK2 complex binds MBP
tightly in the absence of vanadate (Fig. 5) strengthens this
hypothesis. An increased affinity for MBP may be a general feature of
the BPI mutants, since it has been reported that the BPI
MalFG511K2 complex also displays a higher affinity for MBP
than the WT complex (40).
Knowing that MBP stimulates the ATPase activity of MalFGK2,
these results were originally interpreted in terms of an equilibrium between two different conformations of the transporter, an
ATPase-inactive conformation that predominates in the WT and the
ATPase-active conformation induced through interaction with MBP (40).
The BPI mutants were believed to shift this equilibrium in favor of the
active conformation, to a lesser or greater extent, accounting for the
difference in maximal ATP hydrolysis rates between different BPI
mutants (14, 43). Because of induced fit, the active conformation was
expected to have a surface more complementary to MBP than the
ATPase-inactive state and hence have a higher affinity for MBP (40).
With the new knowledge that MBP stimulates ATP hydrolysis by binding
tightly to the transition state conformation of the transport complex,
we now suggest that the BPI conformations may resemble different
intermediates in the pathway of the WT as it approaches the transition
state. These conformational states may not be populated to any great
extent in the cycle of ATP hydrolysis by the WT, providing an
explanation for the failure of MBP to alter noticeably the conformation
of MalFGK2 without vanadate present (Fig. 5).
Alternatively, although less likely, the inactivation caused by MIANS
modification may have prevented attainment of the active conformation.
The increased ability of ATP to protect BPI complexes against MIANS
modification as well as the decreased K0.5 for
ATP hydrolysis by the BPI complexes indicates that the BPI complexes
exhibit an increased affinity for ATP as compared with the WT
complexes. If we are correct in assuming that the BPI complex resembles
an intermediate in the pathway of the WT, then incremental or ratcheted increases in the affinity for both ATP and MBP may be characteristic of
the approach to the transition state for ATP hydrolysis. The affinity
of MalFGK2 for ATP would never exceed the affinity for the
ATP in its transition state conformation, ensuring that ATP hydrolysis
occurs (44). The increased affinity that the BPI complex displays for
ATP could also be a by-product of the specific mutations that induced
the BPI phenotype and unrelated to the normal pathway of activation of
ATPase activity. Analysis of other BPI mutations may resolve this issue.
Recent structural information about ABC proteins aids in the
interpretation of these findings. The crystal structures of two nucleotide-binding subunits of ABC transporters are now available, the
HisP protein from Salmonella typhimurium (45) and the MalK protein from Thermococcus litoralis (46). These two proteins share a similar architecture as expected from the sequence homology, and the nucleotide-binding site is highly exposed on the surface of the
monomer subunit, raising the possibility that residues from another
subunit in the transport complex may contribute to nucleotide binding
and/or hydrolysis (13). Jones and George (13) provide a convincing
theoretical argument in support of a head to tail dimer of HisP in
which the nucleotide-binding sites are buried between the monomers, and
each monomer contributes residues to both nucleotide-binding sites. The
proposed dimer interface, while different from the dimer seen in the
crystal structure of HisP (45), is strikingly similar to a recently published dimer of the RAD50 catalytic domain (RAD50cd), an ABC protein
involved in DNA double strand break repair (25). ATP promotes the
association of two inactive Rad50cd proteins by binding to the
signature motif (or LSGGQ) of one monomer and the P-loop of the
opposite monomer to complete the active site, thereby activating the
ATPase activity (25). The dimer interface seen in the crystal structure
of MalK also positions the nucleotide-binding site between the two
monomers, although the specific residues contributed to the
nucleotide-binding site by the second subunit are not those of the
LSGGQ motif (46). In either case, our data are readily interpretable by
assuming that ATP is bound between the two MalK subunits in
MalFGK2 and that the ATPase activity of the transport complex is regulated by MalK subunit association and dissociation. The
decreased solvent accessibility of the P-loop that is associated with
activation of the MalFGK2 ATPase, either by MBP or by the BPI mutations, may be the result of the nucleotide-binding site becoming more buried in the MalK dimer interface. Activation of the
ATPase activity of the MalK subunit could be achieved if residues on
one subunit are required to complete the nucleotide-binding site on the
second subunit, and the role of the MBP is to bring the two subunits
into closer proximity (Fig. 7). Likewise,
the BPI mutations in MalF may activate the ATPase activity of
MalFGK2 in the absence of MBP by moving the MalK subunits
closer together, explaining why the conformation of the BPI complex
more closely resembles the transition state than the ground state of
the WT transporter. A stable repositioning of key residues in the
second subunit may also account for the increased affinity of the BPI complexes for ATP. Because of the symmetry inherent in a dimer of MalK
or of RAD50cd, association and dissociation would affect both
nucleotide-binding sites equally (Fig. 7) (25), and we propose that it
is this gross conformational change that we are detecting with MIANS at
Cys-40. These data provide the first biochemical evidence in support of
a model of activation of an intact ABC transporter based on subunit
association, as suggested by new structural information. This
interpretation should be treated with caution until further
corroborating evidence is obtained to define the position of the
nucleotide-binding sites within the tetrameric complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
13
atpBC
ilv::Tn10/F' lacIq Tn5) (14) was
transformed with the plasmid pMS421 harboring the
lacIq gene (22) and two additional plasmids,
harboring the malF, malG (pFG23), and malKht
(pSS733) genes under control of Ptrc. The malKht
gene is a variant of malK encoding six histidines at the 5'
end that are used for purification of the transport complex (23). Both
wild-type malF (pFG23) and a malF500 (pFG42)
allele that imparts a binding protein-independent (BPI) phenotype (7) were used. In some experiments, the pSS733 plasmid carrying the malKht allele was replaced with pSSGly, carrying a cysteine
to glycine substitution at a position corresponding to position 40 of
the native MalK protein. Plasmid pSSGly was constructed by cloning the
RsrII-MluI fragment of pGly, containing the
malK C40G mutation ((24) kindly provided by H. Shuman), into
pSS751 (malKht K42N) (23) that had been digested with
RsrII and MluI. Cells were grown as described
previously (12). The total membrane fraction was isolated as described
previously (14) and stored at
70 °C until use.
-D-maltoside to a final concentration of 1%.
Solubilized proteins were bound to a Talon metal affinity column
(CLONTECH) equilibrated with "buffer A" at pH 8 (containing 50 mM NaPi, 300 mM
NaCl, 10% glycerol, and 0.01% dodecyl maltoside). The column was
washed with buffer A at pH 8 and then with buffer A at pH 7. The
transport complexes were eluted from the resin with 100 mM
histidine in buffer A at pH 7. Purified complexes were extensively
dialyzed against "buffer B" (containing 20 mM
NaPi (pH 8), 10% glycerol, 5 mM
MgCl2, and 0.01% dodecyl maltoside) supplemented with 1 mM EDTA.
-32P]ATP (Amersham Pharmacia Biotech) as
described previously (12, 27). ATP hydrolysis activity of complexes in
proteoliposome vesicles was measured at 37 °C in 50 mM
HEPES (pH 7) with 2 mM ATP, 10 mM
MgCl2, 5 µM MBP, and 0.1 mM
maltose using a modification of the technique of Liu et al.
(26).
-mercaptoethanol (MIANS-
-ME), and MIANS-labeled
proteins in buffer B were recorded using an excitation wavelength of
340 nm and an emission range of 360-600 nm. Solvent accessibility was
judged by the ability of acrylamide or
p-xylene-bispyridinium bromide (DPX, Molecular Probes, Inc.) to quench the fluorescence of MIANS. For fluorescence quenching titrations, additions were made from 1 and 5 M acrylamide
stock solutions or from a 0.5 M DPX stock solution.
Quenching was monitored at a constant excitation wavelength of 340 nm
and an emission wavelength corresponding to the emission maximum of the
sample. Fluorescence values were corrected for dilution by performing control titrations with H2O instead of quencher, and the
inner filter effect for acrylamide and DPX at the wavelengths used was negligible. The titration data were used to construct Stern-Volmer plots and KSV values calculated according to
Lakowicz (28). The Stern-Volmer Equation (1) is as follows,
where F0 is the fluorescence intensity in
the absence of quencher; F is the fluorescence intensity in
the presence of quencher; kq is the bimolecular
quenching constant; [Q] is the concentration of the
quencher;
(Eq. 1)
0 is the lifetime of the fluorophore in the
absence of quencher; and KSV is the Stern-Volmer
quenching constant. A plot of F0/F
versus [Q], the extent of quenching as a
function of acrylamide or DPX concentration, yields a line with slope
KSV, a measurement of the accessibility of the
fluorophore to solvent.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Inhibition of MalFGK2 ATPase activity by MIANS
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Fig. 1.
MIANS labeling of Cys-40 and C40G
complexes. WT transport complexes were labeled with MIANS in
detergent solution and analyzed by SDS-PAGE as described under
"Experimental Procedures." A, MIANS fluorescence
associated with transport complex proteins; B, the same gel
stained with Coomassie Blue. Samples were incubated with methanol
(odd-numbered lanes) or MIANS (even-numbered
lanes) as follows: lanes 1 and 2, Cys-40;
lanes 3 and 4, C40G. Arrows indicate
the migration positions of (His)6MalK, MalF, and MalG.
Molecular weight of standards is indicated in
thousands.
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Fig. 2.
Fluorescence spectra of MIANS-labeled
complexes. Transport complexes were labeled with MIANS in
detergent solution as described under "Experimental Procedures."
Fluorescence spectra were recorded using an excitation wavelength of
340 nm and an emission range of 360-600 nm. Fluorescence emission
spectra maxima were as follows: a, 0.1 µM
MIANS- -ME, 443 nm; b, C40G BPI-MIANS, 420 nm;
c, C40G WT-MIANS, 421 nm; d, Cys-40 BPI-MIANS,
421 nm; e, Cys-40 WT-MIANS, 431 nm. The small peak at 385 nm
is the Raman scatter (28). Protein concentrations were 0.1 µM.
-mercaptoethanol in solution. The emission maximum of
MIANS attached to the WT complex was blue-shifted 12 nm from that of
free MIANS-
-ME, whereas the emission maximum of the BPI complex was
blue-shifted 22 nm. These results suggest that the probe is in a more
hydrophobic environment when bound to either protein, and the larger
blue-shift associated with the BPI complex may indicate that the
environment surrounding residue Cys-40 in the nucleotide-binding site
is more hydrophobic in the BPI than in the WT.
1 for free MIANS-
-ME in the
acrylamide experiments), as judged by the decreased slopes of the lines
in the Stern-Volmer plot. In acrylamide quenching experiments, MIANS
bound to the WT complex is significantly more exposed to solvent than
in the BPI complex, with KSV values of 1.94 and
1.41 M
1, respectively (Fig.
3A). Removal of Cys-40 from both complexes negates this
difference, confirming that the assay is reporting on conformational
changes located at the nucleotide-binding site. MIANS bound to these
C40G complexes was even less exposed to solvent than MIANS at Cys-40 in
the BPI transport complex (Fig. 3A). In experiments using
DPX, a larger quenching reagent than acrylamide, MIANS bound to the WT
complex was also significantly more exposed to solvent than in the BPI
complex, with KSV values of 41.5 and 27.3 M
1, respectively. In this case,
the exposure of MIANS on the BPI complex to DPX was the same as that
seen for the complexes containing the C40G substitution (Fig.
3C). These data provide further evidence of a conformational
difference between the WT and BPI complexes which appears to affect the
exposure of residues in the nucleotide-binding pocket to the aqueous
milieu.
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Fig. 3.
Solvent accessibility of Cys-40 and C40G
complexes. Transport complexes containing MalK Cys-40 or C40G
subunits were labeled with MIANS in detergent solution and titrated
with either acrylamide (A and B) or DPX
(C and D) as described under "Experimental
Procedures." ATP was present at 1 mM where indicated. The
Stern-Volmer plots are as follows. A, , 1 µM MIANS-
-ME;
, C40G BPI-MIANS; +, C40G WT-MIANS;
, Cys-40 BPI-MIANS;
, Cys-40 WT-MIANS. B,
,
1 µM MIANS-
-ME;
, Cys-40 BPI-MIANS; +, Cys-40
BPI-MIANS with ATP;
, Cys-40 WT-MIANS;
, Cys-40 WT-MIANS with
ATP. C,
, 1 µM MIANS-
-ME,;
,
C40G BPI-MIANS; +, C40G WT-MIANS;
, Cys-40 BPI-MIANS;
, Cys-40
WT-MIANS. D,
, 1 µM MIANS-
-ME;
, Cys-40 BPI-MIANS; +, Cys-40 BPI-MIANS with ATP;
, Cys-40
WT-MIANS;
, Cys-40 WT-MIANS with ATP. Points are presented as mean
values ± S.D. of titrations performed in triplicate.
View larger version (17K):
[in a new window]
Fig. 4.
Solvent accessibility of vanadate-trapped
complexes. Transport complexes in the presence or absence of MBP
were incubated with or without vanadate as described under
"Experimental Procedures," then labeled with MIANS, and titrated
with acrylamide Stern-Volmer. Plots were constructed, and the resultant
KSV values were as follows: 1,
1 µM MIANS- -ME, 3.50 M
1;
, BPI-MIANS, 1.31 M
1; +, BPI-MIANS with vanadate,
1.33 M
1;
, WT-MIANS, 1.77 M
1;
, WT-MIANS with MBP, 1.74 M
1;
, WT-MIANS with MBP
and vanadate, 1.42 M
1. Points are
presented as mean values ± S.D. of titrations performed in
triplicate.
View larger version (14K):
[in a new window]
Fig. 5.
Binding of MBP to MalFGK2.
WT MalFGK2 in proteoliposomes was incubated with 10 µM maltose, 5 µM MBP, 15 mM
MgCl2, and 5 mM ATP at 37 °C in the presence
or absence of 0.5 mM vanadate, as described previously
(11). Proteoliposomes were then diluted 20-fold into 20 mM
HEPES, 0.1 mM EDTA, collected by centrifugation,
resuspended in HEPES, EDTA, and analyzed by Coomassie staining of
SDS-polyacrylamide gels. The reconstituted BPI transporter was treated
as described for the WT except without maltose and MBP. After washing
to remove unbound vanadate, the BPI proteoliposomes were then incubated
with 5 µM MBP, 10 µM maltose, and 15 mM MgCl2 for an additional 20 min at 37 °C
before being diluted 20-fold into 20 mM HEPES, 0.1 mM EDTA, collected by centrifugation, resuspended in HEPES,
EDTA, and analyzed by SDS-PAGE. Lane 1, molecular weight
standard; lane 2, BPI without vanadate, MBP added;
lane 3, BPI with vanadate, MBP added; lane 4, WT
without vanadate; lane 5, WT with vanadate.
Arrows indicate the migration positions of
(His)6MalK, MalF, MalG, and MBP. Molecular weight of
standards is indicated in thousands.
View larger version (22K):
[in a new window]
Fig. 6.
ATP protection against MIANS
modification. Transport complexes in detergent solution were
preincubated for 15 s with various concentrations of ATP before
addition of MIANS to a final concentration of 20 µM
(A) or 5 µM (B). Incubations were
terminated after 15 min by addition of 1 mM DTT, and the
complexes were assayed for ATP hydrolysis activity: , BPI complexes;
, WT complexes. Values for control incubations with methanol are as
follows: A, BPI, 730 nmol Pi/mg/min; WT, 200 nmol Pi/mg/min. B, BPI, 820 nmol
Pi/mg/min; WT, 70 nmol Pi/mg/min.
ATP protection and kinetic data
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
[in a new window]
Fig. 7.
Model of MalFGK2 ATPase
activation by MalK subunit dimerization. Based on the crystal
structure of the ABC protein RAD50cd (25), the MalK subunits are placed
in a head to tail orientation in the MalFGK2 complex with
the nucleotide-binding sites located at the dimer interface. Both MalK
subunits contribute residues to each nucleotide-binding site, providing
a mechanism for regulating ATPase activity via MalK subunit association
and dissociation within the transport complex. Our data suggest that,
in the wild type, the MalK subunits are positioned apart, preventing
ATP hydrolysis in the absence of MBP, and the residues in the
nucleotide-binding site are relatively more accessible to solvent. MBP
activates the ATPase activity of the wild type by moving the MalK
subunits closer together, rendering the nucleotide-binding sites less
accessible to solvent. In a binding protein-independent complex
containing the MalF500 mutations, the MalK subunits are positioned
closer together, allowing ATP hydrolysis to occur in the absence of
MBP.
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ACKNOWLEDGEMENTS |
---|
We thank H. A. Shuman for kindly providing the Malk C40G mutant, Steen Pedersen for use of the ISS PC1 photon counting spectro fluorometer, and Jue Chen and Fred Gimble for reading the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by United States Public Health Service Grant GM49261 and Robert A. Welch Foundation Grant Q-1391.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.
Supported in part by United States Public Health Service
Predoctoral Training Grant GM08231.
§ To whom correspondence and reprint requests should be addressed: Dept. of Molecular Virology and Microbiology, MS: BCM 280, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-4552; Fax: 713-798-7375; E-mail: davidson@bcm.tmc.edu.
Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M011686200
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ABBREVIATIONS |
---|
The abbreviations used are:
ABC, ATP-binding
cassette;
MBP, maltose-binding protein;
BPI, binding
protein-independent;
WT, wild-type;
MIANS, 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid;
dodecyl maltoside, n-dodecyl -D-maltoside;
DTT, dithiothreitol;
-ME,
-mercaptoethanol;
DPX, p-xylene-bispyridinium
bromide;
PAGE, polyacrylamide gel electrophoresis;
NEM, N-ethylmaleimide.
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