(Received for publication, October 2, 1995; and in revised form, December 1, 1995)
From the
Maltose transport across the cytoplasmic membrane of Escherichia coli is catalyzed by a periplasmic binding protein-dependent transport system and energized by ATP. The maltose system, a member of the ATP-binding cassette or ABC transport family, contains two copies of an ATP-binding protein in a complex with two integral membrane proteins. ATP hydrolysis by the transport complex can be assayed following reconstitution into proteoliposomes in the presence of maltose binding protein and maltose. Mutations in the transport complex that permit binding protein-independent transport render ATP hydrolysis constitutive so that hydrolysis can also be assayed with the transport complex in detergent solution. We have used both of these systems to study the role of two ATP binding sites in ATP hydrolysis. We found that both the wild-type and the binding protein-independent systems hydrolyzed ATP with positive cooperativity, suggesting that the two ATP binding sites interact. Vanadate inhibited the ATPase activity of the transport complex with 50% inhibition occurring at 10 µM vanadate. In detergent solution, the degree of cooperativity in the binding protein-independent complex decreased with increasing pH. The loss of cooperativity was accompanied by a decrease in ATPase activity and a decrease in sensitivity to vanadate. Because reconstitution of the complex into a lipid bilayer prevented the loss of cooperativity, we expect that ATP hydrolysis is cooperative in vivo. The mutations leading to binding protein-independent transport do not significantly alter the affinity, cooperativity, vanadate sensitivity, or substrate specificity of the ATP binding sites during hydrolysis. These results justify the use of the binding protein-independent system to investigate the mechanism of transport and hydrolysis.
The maltose transport system of Escherichia coli is one
of a class of periplasmic binding protein-dependent systems that
function to transport nutrients across the cytoplasmic membrane against
a concentration gradient. These systems are part of a larger family of
ATP-dependent transporters termed ATP binding cassette or ABC
proteins(1) . Members of the family are involved in many
diverse functions, including ion transport(2) , protein and
peptide transport(3, 4) , drug
transport(5, 6) , and polysaccharide
transport(7, 8) . A prominent feature of this family
is its multi-domain, or multi-subunit structure, which includes two
hydrophobic subunits capable of spanning the membrane in -helical
conformation and two hydrophilic subunits capable of binding ATP. The
maltose transport complex consists of MalF, MalG, and two copies of
MalK, an ATP-binding protein(9, 10) . Reconstitution
of the purified transport complex (MalFGK
) into
proteoliposome vesicles demonstrated that these three proteins, the
periplasmic maltose binding protein (MBP), (
)and ATP are
necessary and sufficient for maltose transport(9) .
In
proteoliposomes, MalFGK requires both maltose and MBP to
hydrolyze ATP(11) . However, mutations in malF and malG, isolated as a consequence of their ability to support
maltose transport in the absence of the MBP(12) , permitted ATP
hydrolysis to occur in the absence of maltose and MBP(11) .
These experiments formed the basis for the current model for transport (Fig. 1A), in which the interaction of the maltose-MBP
complex with the transport complex shifts the conformation of the
transport complex from an inactive form (I), which cannot hydrolyze
ATP, to an active form (II), which can hydrolyze ATP and therefore
transport maltose (III). This conformational change coordinates the
appearance of maltose in the periplasm with ATP hydrolysis. The
mutations that render transport binding protein independent stabilize
the same active form (II) (Fig. 1B). This model
predicts that during ATP turnover, the wild-type and mutant complexes
are functionally identical. One goal of the present work is to test
this hypothesis by characterizing hydrolysis in both the wild-type and
the mutant systems. If they are similar, then we expect that the
mutations have stabilized a normal intermediate in the reaction pathway
and that use of the binding protein-independent mutants will facilitate
the elucidation of the mechanism of ATP-dependent translocation. Unlike
the wild-type transport complex, the mutant complex will hydrolyze ATP
while in detergent solution, simplifying many biochemical and
biophysical measurements.
Figure 1: Model for maltose transport. A, in the wild type, interaction with the periplasmic maltose-MBP complex shifts the conformation of the membrane transport complex from an inactive form (I), which cannot hydrolyze ATP, to an active form (II), which can hydrolyze ATP. The hydrolysis of ATP is coupled to a second conformational change that results in vectorial transport (III). B, mutations in MalF or MalG, which permit binding protein-independent transport, stabilize the active conformation (II) in the absence of MBP, allowing constitutive ATP hydrolysis and cyclic conformational changes that result in transport (III) (modified from (11) ).
Little is known about the molecular mechanism of ATP hydrolysis and transport in this family. The conservation of two nucleotide binding domains in this family of proteins suggests that both will be essential for transport activity. A second goal of this work is to examine the kinetics of ATP hydrolysis to look for interactions between the two ATP binding sites in the maltose transport complex. We find that ATP is hydrolyzed with positive cooperativity.
Freshly transformed cells were grown and induced for overexpression as described(9) . Cell envelope fractions were prepared as described (11) and stored at -70 °C until use.
where v = velocity, [S]
= [ATP], V = maximum
velocity, K
or K
=
(the concentration of S where v =
V
), and n = the Hill
coefficient, or the number of substrate binding sites per molecule of
enzyme. Chi square analysis was used to determine which equation best
fit the data(16) .
The ability of the ATPase to hydrolyze
other nucleotides was tested as described above using sodium acetate
buffer at pH 5 for the detergent-soluble form of the enzyme and
Bis-Tris at pH 6 for the reconstituted systems, except that unlabeled
substrates were employed and P was determined using a
microtiter plate method(17) . Absorbance was read at 650 nm.
Figure 2:
pH dependence of ATP hydrolysis. ATP
hydrolysis by the wild-type and binding protein-independent complexes
was measured as a function of pH of the assay medium using 1 mM ATP (see ``Experimental Procedures''). To detect any
indirect effects of the buffers on activity, experiments were designed
such that the pH range tested with each buffer overlapped with the
range of the next buffer. Buffers used were acetate (pH 4-5),
succinate (pH 5-6), Mes (pH 6-6.5), Mops (pH
6.5-7.5), Hepes (pH 7.5-8.5), Tris (pH 8.5-9.5),
ethanolamine (pH 9.5-pH 10), and Caps (pH 10-11). ,
wild-type complex in proteoliposome vesicles;
, MalF500 complex in
proteoliposome vesicles; &cjs3648;, MalF500 complex in
detergent-soluble form.
The ATPase activity of the
MalF500GK complex was also assayed in detergent solution
following solubilization with dodecyl maltoside. Under these
conditions, the complex displayed a more acidic pH optimum, pH 5.5. The
activity of the detergent-soluble form of the MalF500GK
complex was lower than that of the reconstituted form, except at
low pH where the activities were comparable. In experiments where the
detergent-soluble and reconstituted forms of the complex were incubated
at room temperature for 2 h at pH levels from 4 to 11, then neutralized
and assayed at the optimum pH levels irreversible inactivation occurred
only at pH 4.
Figure 3:
Kinetics of ATP hydrolysis by
MalF500GK in detergent solution. The binding
protein-independent (MalF500) transport complex was solubilized from
the membrane with dodecyl maltoside, and activity was measured as a
function of ATP concentration (see ``Experimental
Procedures''). Two experiments taken from the data summarized in Table 1are shown. Curves were generated by fitting the data to
the Michaelis-Menten equation (dashed line) and to the Hill
equation (solid line) (see ``Experimental
Procedures''). Insets represent enlargements of the
figures. A, 50 mM sodium acetate, pH 5.
Michaelis-Menten: V
= 527 ± 40, K
= 27.0 ± 6; Hill: V
= 451 ± 9, K
= 18.8 ± 0.8, n
= 1.9
± 0.1. B, 50 mM Tris-HCl, pH 8.
Michaelis-Menten: V
= 203 ± 6, K
= 15.7 ± 1.7; Hill: V
= 192 ± 7, K
= 13.8 ± 1.4, n
= 1.2
± 0.1.
The
cooperativity in hydrolysis is less apparent at pH 8, with n = 1.2 ± 0.1 (Fig. 3B). To test the validity of including an
additional term (n) in the fitting equation, an F test on
, a measure of goodness of fit was
performed(16) . At pH 5, the fit to the Hill equation was far
superior to that of the Michaelis-Menten equation (p <
0.001). However, at pH 8 the Michaelis-Menten equation with just two
variables, V
and K
, was
adequate to describe the data (p > 0.25). This difference
cannot be explained by the presence of a contaminating activity since
the detergent-soluble membrane fraction isolated from the host strain
without plasmids had little detectable ATPase activity when assayed at
100 µM ATP in this pH range (data not shown).
Table 1summarizes several more experiments of the type shown
in Fig. 3in which the MalF500GK complex was assayed
in detergent-soluble form at different pH levels. The half-saturation
constant, K
, is approximately 20 µM and is not greatly affected by changes in pH. As the data in Fig. 3indicated, n
decreases with
increasing pH.
Figure 4:
Kinetics of ATP hydrolysis in
proteoliposome vesicles. ATPase activity was measured as a function of
ATP following reconstitution of the wild-type and MalF500 transport
complexes into proteoliposome vesicles as described under
``Experimental Procedures.'' Two experiments taken from the
data summarized in Table 2are shown. Curves were generated by
fitting the data to the Michaelis-Menten equation (dashed
line) and to the Hill equation (solid line). A,
MalF500GK complex assayed in 20 mM potassium
P
, pH 6. Michaelis-Menten: V
= 1920 ± 220, K
= 200 ± 43; Hill: V
= 1280 ± 50, K
= 88
± 6, n
= 1.7 ± 0.1. B, wild-type complex assayed in 20 mM potassium
P
, pH 6. Michaelis-Menten: V
= 34.3 ± 2.3, K
= 168 ± 25; Hill: V
= 27.0 ± 1.7, K
= 102
± 13, n
= 1.4 ±
0.1.
Figure 5:
Inhibition of ATPase activity by vanadate.
ATP hydrolysis by MalF500GK in dodecyl maltoside solution
was measured in the presence of increasing concentrations of vanadate.
Assays were buffered at pH 5 using 50 mM sodium acetate.
, 100 µM ATP;
, 10 µM ATP.
Figure 6:
pH
dependence of vanadate inhibition and Hill coefficient of
MalF500GK in detergent-soluble form. Percent inhibition,
, was calculated from the differences in the rates of hydrolysis
at 100 µM ATP in the presence and absence of 100
µM vanadate. Values for the Hill coefficient, n
(
), were taken from Table 1. Curves
were generated by fitting data to the rate equation described in the
text and used to calculate the pK
values
for the transitions.
where y = percentage inhibition or n,
the Hill coefficient, H the proton concentration, K
the ionization constant, and A
and A
represent the upper and lower
limits of y for the protonated and unprotonated forms,
respectively. A
, A
, and K
were determined by least squares analysis,
except in the analysis of the % inhibition, where A
was fixed at 100%. The pK
values of the
transitions were estimated to be 5.2 ± 0.1 for inhibition by
vanadate and 5.6 ± 0.1 for cooperativity (n
). In contrast, in the reconstituted system,
where cooperativity was not a function of pH, vanadate inhibited
strongly at all pHs; in assays with 1 mM ATP, 100 µM vanadate inhibited 80-90% of the ATPase activity. The
wild-type transport complex, assayed at pH 6 in potassium P
buffer, was also inhibited by vanadate (not shown).
The conservation of two nucleotide-binding domains in the ABC transport family suggests that both are central to function, yet little is known about the role of these two sites in ATP hydrolysis and transport. To address this question, we examined the dependence of ATP hydrolysis on ATP concentration in the maltose transport system, and we discovered that ATP was hydrolyzed with positive cooperativity. Cooperativity likely results from interactions between the two MalK subunits, with the binding of ATP to the first subunit enhancing the affinity of binding to the second subunit. An alternative explanation, that ATP-induced oligomerization resulted in cooperativity, is unlikely because, in detergent solution, activity was independent of protein concentration. A tight binding site for nucleotides would lead to positive cooperativity only when the concentration of enzyme was high relative to the substrate concentration, which was not true in these experiments. Finally, a contaminating ATPase activity could generate negative but not positive cooperativity.
The maltose system is the first ABC transporter in which cooperativity has been detected. This is of particular interest because kinetic analyses have been carried out on the ABC protein MDR in several different laboratories, but no positive cooperativity has been detected (21, 27, 28, 29) . It is not clear if cooperativity was lost in vitro in the MDR system or if the two systems use different mechanisms to hydrolyze ATP despite their structural similarities.
What is the physiological significance of positive cooperativity in this system? It is possible that positive cooperativity may play a key role in the reaction mechanism. The conformational changes required to facilitate the binding of ATP to the second site may be critical in coupling ATP hydrolysis to the transport event. In other transport ATPases, two mechanisms have been proposed to explain how ATP might stabilize the conformational changes required for transport. These include phosphorylation of the protein, as in the P-type ATPases, or tight binding of nucleotide, as in the F-type ATPases(23) . To date, no evidence for either of these mechanisms has been found in any ABC protein.
Cooperativity in ATP
hydrolysis was a function of pH in the detergent-soluble form of the
MalF500GK complex but not in the reconstituted system.
Since prior exposure to buffers in the pH range from 11 to 4.5 did not
result in any irreversible inhibition of ATPase activity, this
transition must relate to changes in the protonation state of the
soluble transport complex. A critical residue(s) in the
detergent-soluble form of the complex must be protonated for positive
cooperativity to occur. In the reconstituted system, where there is no
effect of pH, it can be argued that protein-lipid interactions either
shift the pK
of the residue(s) to more alkaline pH
or, alternatively, stabilize quaternary interactions in the absence of
the proton(s).
The pH dependence of ATP hydrolysis (Fig. 2)
supports the hypothesis that a critical change is taking place in the
detergent-soluble form of the complex near pH 5.5. Following
reconstitution of the MalF500GK complex into
proteoliposomes, the pH dependence curve has a typical bell shape with
a maximum at pH 8. The detergent-soluble form of the complex has a more
unusual pH profile with a maximum at pH 5.5. Activity of the soluble
form is substantially lower than the membrane-associated form at high
pH, but it begins to rise at pH 6, ultimately matching the activity of
the reconstituted form at pH 5. We interpret these curves to mean that
protonation of the soluble form of the complex results in an increase
in the efficiency of ATP hydrolysis as cooperative interactions are
restored. Hence, we suggest that the form of the enzyme present at pH 5
in detergent solution is more representative of the actively
transporting species in vivo, despite the fact that the pH in
the cytoplasm is near neutrality(24) . As discussed above, the
presence of the membrane appears to prevent the loss of cooperativity
and activity at neutral pH.
The susceptibility of the detergent-soluble form of the complex to the inhibitor vanadate also changes dramatically as a function of pH. ATP hydrolysis is inhibited at pH 5 but not at pH 8. The ionic state of vanadate does not change between pH 5 and pH 8(25) . The correlation between vanadate sensitivity and cooperativity in the detergent-soluble form (Fig. 6) strengthens the argument that the detergent-soluble form is undergoing a significant transformation near pH 5.5. This correlation is also seen in the reconstituted system, where both inhibition and cooperativity were seen at all pHs tested, and in the isolated MalK protein, where ATP hydrolysis followed Michaelis-Menten kinetics and was insensitive to vanadate(20) . Deprotonation of the detergent-soluble form of the transport complex appears to alter the conformation of the ATP binding site such that vanadate can no longer inhibit and the two sites can no longer interact. While this conformational change, detected as a function of pH while studying the binding-protein-independent system in detergent-soluble form, may not represent a physiologically significant transformation, we are investigating the possibility that a similar conformational change may play an important role in the reaction cycle of the wild-type transport system under more physiological conditions.
Vanadate inhibits MDR by trapping ADP in one of the two ATP binding sites(26) . Vanadate presumably acts in concert with magnesium and ADP to form a transition state analogue that is tightly bound by the enzyme. While the correlation between vanadate sensitivity and cooperativity found in our work could be purely coincidental, with two separate protonation events mediating the two changes, a far more interesting possibility is that this relationship reflects an integral part of the molecular mechanism of hydrolysis itself. For example, the ability to bind the transition state analogue tightly may be an essential feature of the signaling event between the two MalK subunits that results in cooperativity. Further experiments will be required to investigate this possibility.
On the basis of the work in this paper, it appears that the use of the binding protein-independent (malF500) mutation is not significantly altering the characteristics of the ATPase in the reconstituted system, including the half-saturation constant, positive cooperativity, susceptibility to vanadate, and substrate specificity. These data support our model (Fig. 1) in which we propose that the MalF500 mutation stabilizes the complex in an active conformation (II) similar to the wild-type complex when it is stimulated by MBP. Thus, prudent use of the MalF500 system as well as the wild-type system should provide valuable insight into the mechanism of action of the transport complex. Using the manifestation of cooperativity as a guide, the best system for studying ATP hydrolysis is the reconstituted system that displayed cooperativity at all pHs. Where indicated, use of the detergent-soluble form of the complex at acid pH should also yield physiologically significant results.