From the Institute of Biochemistry, Biocenter, J. W. Goethe-University, Marie-Curie-Str. 9, D-60439 Frankfurt a.M., Germany
Received for publication, February 4, 2003 , and in revised form, May 3, 2003.
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ABSTRACT |
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INTRODUCTION |
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Half-size ABC transporters, like the heterodimeric TAP and homodimeric Mdl1p, have a common molecular architecture consisting of two polytopic transmembrane domains (TMD) and two nucleotide-binding domains (NBD). The transmembrane domains interact with the substrates and form the substrate translocation pore across the membrane. The TMDs generally share little homology (5), probably caused by the broad substrate spectrum of the ABC transporter family. Binding and hydrolysis of nucleotides drive the transport process by transducing conformational changes from the NBDs to the TMDs. The similarity of different NBDs is significantly higher compared with the TMDs, suggesting that even in transporters of unrelated function the structure and function of the NBDs be highly conserved. Each NBD contains a highly conserved Walker A and Walker B motif (6) characteristic of ATP-binding P-loop proteins, as well as the C-loop motif (LSGGQ) unique to ABC proteins, which is also known as the ABC signature motif. The crystal structures of bacterial ABC transporters (e.g. MsbA, BtuCD) and of isolated NBDs (e.g. HisP, MalK, MJ1276, and TAP1) show a consensus fold for the monomer (714). This fold shows an L-shaped molecule with two arms, one including the ATP-binding domain containing the Walker A and B motifs, and the other including the ABC signature motif (7). The crystal structures of several NBD dimers have been solved, but they differ significantly in the manner the monomers are associated (15, 16). The crystal structure of the ATP-bound Rad50 dimer, a DNA repair enzyme that shares homology with the NBDs of ABC proteins is assumed to be the physiologically relevant dimer. This dimer contains two nucleotides, which are clamped at the interface between two monomers causing them to dimerize in a head-to-tail manner (17). The ATP molecules are sandwiched between the Walker A and B motifs from one monomer and the C-loop from the other monomer. This proposed functional dimer configuration was also found in the crystal structure of a mutant NBD from Methanococcus jannaschii MJ0796 (14) and in the NBD of the full-length bacterial vitamin B12 transporter BtuCD (13). Biochemical data on NBDs like MalK or of P-gp further confirmed the involvement of the C-loop in ATP binding (18, 19). The dimer is generally regarded as the ATPase active form of ABC transporters, but it remains controversial at which point of the transport cycle the dimer is formed and how both NBDs cooperate. Whether the motor domains work as equivalent modules, hydrolyzing both ATP synchronously, sequentially or alternatively is still under discussion. It is also not clarified how ATP hydrolysis powers the substrate translocation. Based on the P-gp ATPase cycle, it was suggested that hydrolysis of one ATP provides energy for the translocation of the substrate whereas hydrolysis of the ATP in the second binding pocket might have regulatory functions, possibly returning the complex to the starting point of the ATPase cycle (20, 21).
To obtain insight into the mechanism of the NBDs, we expressed and purified the NBD of Mdl1p. Either by incubation with orthovanadate or beryllium fluoride, or by mutagenesis of the NBD, catalytic transition states were trapped and the nucleotide composition of these states was analyzed. Based on these results, we propose a new model for the ATPase cycle combining structure and function of the ABC dimer.
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EXPERIMENTAL PROCEDURES |
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Purification of Mdl1p-NBDThe cell pellet was resuspended in lysis buffer (PBS: 10 mM Na2HPO4/NaH2PO4, pH 7.6, 150 mM NaCl, 5 mM KCl), 20 mM imidazole containing 1 mM phenylmethylsulfonylfluoride, 1% lysozyme, and 510 units of benzonase (Merck) and disrupted by French press treatment at 1,000 p.s.i. After centrifugation for 30 min at 100,000 x g, the supernatant was applied on a Ni-IDA column (Amersham Biosciences) preequilibrated in PBS supplemented with 20 mM imidazole. The column was washed with step gradients of 80 and 100 mM imidazole in PBS, and the protein was eluted with 250 mM imidazole. Fractions containing the NBD were pooled, concentrated by Centricon 10 (Millipore), and applied to a Superdex 200 HR 26/60 prep grade gel filtration column (Amersham Biosciences) in 20 mM Tris, pH 8.0, 100 mM NaCl. The NBD eluted with an apparent molecular weight of 33 kDa corresponding to the size of the monomer. Fractions were concentrated to 10 mg/ml and stored at 4 °C. Wild-type and mutant proteins were purified to greater than 99% homogeneity as assessed by Coomassie-stained protein on SDS-PAGE (15%) and MALDI-TOF MS.
8-Azido-ATP PhotolabelingAfter preincubation of the
purified NBD (0.3 µM) with different concentrations of
nucleoside tri/diphosphates for 5 min in binding buffer (20 mM
Tris, pH 8.0, 100 mM NaCl, 5 mM MgCl2, 1
mM MnCl2) on ice, 8-azido-[-32P]ATP
was added to a final concentration of 0.5 µM and incubation was
continued on ice for 5 min. Samples were irradiated by UV (254 nm) for 5 min,
directly resuspended in SDS loading buffer, and separated by 15% SDS-PAGE. The
gel was dried and quantified by phosphor imaging. The data were fitted to the
Hill equation y = (axn/(bn +
xn)), resulting both for ATP and ADP in a best fit
(R2 = 0.98) with a Hill coefficient (n) of 1.
ATPase AssayATPase activities were measured by the
malachite green assay as described previously
(22). The ATPase assay was
performed in 20 mM Tris, pH 9.0, 100 mM NaCl and 15
mM MgCl2 with 0150 µM NBD for
either 2 min at 16 °C (high NBD concentrations) or 4 min at 30 °C. The
reaction was started by the addition of ATP to final concentrations of 0 to 10
mM. Data were fitted to the Hill equation y =
(axn/(bn + xn)).
The ATPase activity of the E599Q mutant was determined using radiolabeled ATP.
Shortly, E599Q NBD (250 µM) was incubated in 20 mM
Tris, pH 9.0, 100 mM NaCl, and 15 mM MgCl2 at
30 °C. The reaction was started by addition of 2 mM MgATP
supplemented with 0.04 µM [-32P]ATP (4500
Ci/mmol, ICN). Samples were taken at different time points (0200 min),
separated by thin layer chromatography (see below) and the rate of released
radioactive phosphate was quantified by a phosphorimager. As a negative
control, spontaneous ATP hydrolysis was tested in the presence of
heat-inactivated NBD.
Dimerization AssaysBefore use, orthovanadate stock solution adjusted to pH 8.0 was boiled for 5 min to break polymeric species (23). Before use, beryllium fluoride solution was prepared by mixing BeCl2 and NaF in a ratio of 1:100. For non-radioactive assays, purified wild-type NBD (30 µM) was incubated with different concentrations of beryllium fluoride (BeFx) or orthovanadate in the presence or absence of nucleotides and nucleotide analogues for 5 min at 30 °C in binding buffer. The mutant NBD (E599Q, 30 µM) was incubated for 5 min under non-hydrolyzing conditions (in the absence of Mg2+ and on ice) or ATP hydrolysis conditions (in the presence of 5 mM Mg2+ and at 30 °C) with different concentrations of nucleotides and nucleotide analogues. The samples were applied to a Superdex 75 PC 3.2 (Amersham Biosciences) gel filtration column at a flow rate of 50 µl/min at 4 °C in 20 mM, Tris pH 8.0, 150 mM NaCl, and 5 mM MgCl2 if indicated.
Determination of the Nucleotide StoichiometryRadioactive
trapping experiments, were performed in the same buffer as non-radioactive
trapping experiments, and separated by gel filtration. Fractions were analyzed
by -counting, thin layer chromatography, and for protein concentration
by the BCA assay (Pierce). Over the range of protein concentrations used, the
A280 measured with the UV detector of the SMART system
(Amersham Biosciences) was linearly related to the protein concentration
determined by the BCA assay. In particular, wild-type NBD (250
µM) was incubated with 500 µM MgATP supplemented
with 0.1 µM of either [
-32P]ATP or
[
-32P]ATP (4500 Ci/mmol, ICN) in the presence of
BeFx (500 µM) for 5 min at 30 °C. The E599Q
mutant (250 µM) was incubated for 5 min in the absence of
Mg2+ and on ice. To follow one to two turnovers of the
E599Q mutant, 250 µM NBD were incubated for prolonged time
periods (0500 min) at 30 °C with limiting concentration of MgATP
(250500 µM) and tracer amounts of radioactive ATP. To
test whether ADP is incorporated in the dimer at a high ADP concentration, the
E599Q mutant was incubated with 500 µM ADP and 0.1
µM [
-32P]ADP in the presence of various
concentration of unlabeled ATP (0, 5, 50, 100, and 500 µM) for
10 min at 30 °C. Alternatively, incorporation of ADP under limiting
concentrations of MgATP was tested by incubating the E599Q mutant (250
µM) with MgATP (250500 µM) supplemented
with tracer amounts of [
-32P]ADP at 30 °C for 400 min.
The [
-32P]ADP was formed by incubation of
[
-32P]ATP with hexokinase (Sigma Aldrich) according to the
manufacturer's instructions, separated from hexokinase by Centricon 10
centrifugation, and full conversion to [
-32P]ADP was
confirmed by TLC.
Thin Layer ChromatographyThe nucleotide composition of the
dimer was analyzed by TLC. Immediately after gel filtration the fraction
containing the dimer was incubated with 15 mM EDTA for 30 min and
precipitated with trichloroacetic acid (10% w/v) for 30 min at 4 °C. After
centrifugation at 20,000 x g for 5 min, the sample was
neutralized with 0.5 M KHCO3, pH 8.3 and applied onto
polyethylenimine cellulose plates (Merck) in 2 M formate and 250
mM LiCl. [-32P]ATP and
[
-32P]ATP treated with hexokinase were used as
references.
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RESULTS |
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The Active Form of NBD Is a DimerIt is postulated that the interaction of both NBDs plays a central role in the catalytic cycle of ABC transporters (see review in Ref. 24). So far, gel filtration experiments with isolated NBDs (e.g. HisP or NBDs of P-gp) could not demonstrate ATP-dependent dimerization, independent of the presence of nucleotides or non-hydrolyzable analogues such as AMP-PNP (25, 26). The wild-type NBD of Mdl1p also behaved as a monomer in gel filtration studies independent of the presence of nucleotides. To analyze intermediate states of the ATPase cycle, we tried to trap the NBD in the conformation directly after ATP hydrolysis using orthovanadate (Vi) or beryllium fluoride (BeFx). As shown on myosin x-ray structures the vanadate-inhibited complex resembles a post-hydrolysis state while the complex trapped by BeFx represents a pre-hydrolysis state (27). After 5 min of preincubation of the NBD with BeFx in the presence of MgATP at 30 °C, a stable dimer corresponding to a molecular mass of 66 kDa was observed by gel filtration even in the absence of MgATP and BeFx in the mobile phase (Fig. 4A). The formation of the dimer was dependent on the concentration of BeFx. At a concentration of 1 mM BeFx almost 80% of the protein was detected as a dimer. The same result was observed with orthovanadate (Fig. 4B). Importantly, neither preincubation of the NBD with MgADP or MgAMP-PNP in the presence of BeFx/vanadate at 30 °C, nor incubation with MgATP and BeFx/vanadate at 4 °C induced dimerization of NBD. Omitting Mg2+ from the reaction inhibited the formation of dimers. This demonstrates that ATP hydrolysis is essential for the formation of the BeFx/vanadate-stabilized dimer.
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ATP-dependent Dimerization of the E599Q Mutant NBD Exchange
of the conserved glutamate downstream of the Walker B motif (E552Q/E1197Q) of
mouse P-gp produced an ABC transporter with severely impaired biological
activity and no substrate-stimulated ATPase activity
(28). Equivalent mutation of
the archaeal NBDs of MJ0796 and MJ1267 (E171Q/E179Q) induced an ATP-dependent
dimerization of the NBDs (29).
We introduced an equivalent mutation in the conserved glutamate (E599Q) in the
NBD of Mdl1p, which was expressed and purified in equal amounts as the
wild-type protein (Fig. 1). The
mutant NBD bound ATP to the same extent as the wild-type as analyzed by
8-azido-ATP photolabeling experiments (data not shown). As expected, no
steady-state ATP hydrolysis was observed by measuring the release of
Pi using the malachite green assay (data not shown). To assay the
ability of the mutant to form nucleotide-dependent dimers, we incubated the
mutant NBD with MgATP on ice for 5 min. Gel filtration experiments showed that
MgATP induced dimerization in a concentration-dependent manner
(Fig. 5). The E599Q NBD dimer
was stable during gel filtration even without nucleotides in the mobile phase,
indicating that the ATP-induced dimer only slowly dissociates. Importantly,
ADP did not induce dimerization, and the addition of ADP to the monomers
impaired ATP-dependent dimer formation. Addition of Mg2+
(05 mM) did not influence the dimerization. Furthermore,
MgAMP-PNP or MgATPS could bind to the wild-type and mutant NBD of Mdl1p
to the same extent as MgATP, but did neither induce dimerization of the
wild-type NBD nor of the E599Q mutant (data not shown). Based on these results
we can conclude that presence of ATP is absolutely required for the formation
of both wild-type and mutant dimer.
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Nucleotide Composition of the DimerTo analyze the ATP/ADP
composition of the nucleotide-induced dimer, wild-type NBD was incubated with
tracer amount of either [-32P]ATP or
[
-32P]ATP under BeFx-trapping conditions and
applied to the gel filtration column. Radioactive nucleotides exclusively
coeluted with the dimer, whereas no radioactive nucleotides were observed in
fractions corresponding to the monomer
(Fig. 6A). The amount
of [
-32P]ATP bound per wild-type dimer was determined and a
stoichiometry of two nucleotides per dimer was obtained
(Fig. 6A, open
circles). Trapping the NBD by BeFx in the presence of
[
-32P]ATP resulted in a dimer with almost no radioactive
nucleotides incorporated (Fig.
6A, filled circles,
Table I). These results
demonstrate that two ATP molecules are already hydrolyzed in the
BcFx-trapped wild-type dimer and the
[
-32P]i is released from the complex. This was
confirmed by TLC analysis (Fig.
6A). Under the same conditions, incorporation of
[
-32P]ADP into the dimer was not observed, even in the
presence of different concentrations of ATP (data not shown).
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In experiments with the E599Q mutant, performed at 4 °C and in the
absence of Mg2+, the nucleotides exclusively coeluted
with the dimer as well. In this experiment, the amount of both
[-32P]ATP and [
-32P]ATP bound per dimer
was equal and a stoichiometry of two ATP molecules per dimer was obtained
(Fig. 6B,
Table I). TLC clearly
demonstrated that ATP and not ADP and Pi were incorporated into the
dimer. As mentioned above, incubation with non-hydrolyzable ATP analogues did
not trigger dimer formation.
Our data from both the BeFx-trapped wild-type NBD and the E599Q mutant suggest that during the ATP hydrolysis cycle at least two different intermediate states can be isolated. Importantly, both states contain two nucleotides. In one state the nucleotides are bound as ATP as shown for the E599Q mutant. In the other state both nucleotides have been hydrolyzed to ADP and Pi as demonstrated for the trapped wild-type NBD. This suggests that ATP binding on both NBDs induces formation of the dimer and that after hydrolysis of both ATP molecules to ADP, the dimeric complex dissociates and ADP is released.
Hydrolysis Cycle of the E599Q MutantAn important remaining
question is how the ATPs are hydrolyzed during one cycle. Using radiolabeled
ATP, it was determined that the E599Q mutant, at 30 °C and in the presence
of Mg2+ was still able to hydrolyze ATP with a turnover
rate of 0.5 ATP per hour (data not shown). The ATPase activity of the E599Q
mutant was 3000-fold reduced compared with the wild-type. The amount of
released phosphate in the radioactive ATPase assay increased linearly over
several hours, indicating that the E599Q mutant can make multiple turnovers.
The strongly reduced turnover rate gave us a tool to examine the hydrolysis
cycle in more detail. When the dimer was incubated with limiting amounts of
ATP at 30 °C in the presence of Mg2+ in order to
allow only one or two ATP hydrolysis turnovers, the gel filtration analysis of
the E599Q mutant showed that, over time, the amount of dimeric NBD decreases
and the amount of monomeric NBD increases (e.g. after 400 min,
Fig. 6, C and
D, left panel). The nucleotide composition of
the remaining dimer was analyzed, and was found to contain one ATP and one ADP
(Fig. 6C,
Table I). TLC analysis of the
dimer confirmed an intermediate state containing equal amount of ATP and ADP
(Fig. 6C, middle
panel). The dimers isolated after prolonged incubation times at 30 °C
(Fig. 6D, left
panel) reached a stoichiometry of one ATP and one ADP
(Fig. 6D, right
panel), suggesting that this is a stable intermediate state, which can be
detected under limiting ATP conditions. To exclude that the ADP found in the
dimer is derived from ADP rebinding from the solution, ADP incorporation was
followed under exactly the same conditions using [-32P]ADP
as a tracer. Under these conditions no incorporation of
[
-32P]ADP was detected
(Table I). This demonstrated
that the dimer-bound ADP is derived from hydrolysis of the first ATP. The
intermediate state suggests that the ATP hydrolysis occurs by a sequential
mechanism, and that in the E599Q mutant hydrolysis of the second ATP is slower
than hydrolysis of the first.
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DISCUSSION |
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Mdl1p has recently been identified as an intracellular homodimeric half-size ABC transporter localized in the inner mitochondrial membrane of S. cerevisiae (3). The peptide transporter Mdl1p shares functional and structural homology to the heterodimeric TAP. We are interested in the mechanism of peptide transport across membranes, and especially how ATP drives this transport. As the Mdl1p transporter is proposed to work as a homodimer, we chose the NBD of Mdl1p as a model. This is the first report with mechanistic data on Mdl1p, and no in vitro data are presently available for the full-length Mdl1p. The C-terminal domain (amino acids 427695) corresponding to the NBD was overexpressed as soluble protein in E. coli and purified to homogeneity. The purified NBD was active in ATP binding and hydrolysis. The turnover number of the Mdl1p-NBD (25 min1) is comparable to the values obtained for other isolated NBDs (e.g. MJ0796, 12 min1, Ref. 29, CFTR, 6.7 min1, Ref. 34). In general, isolated NBDs show much lower turnover numbers than the full-length transporters, and no substrate stimulation (26). The ATPase activity of isolated Mdl1p-NBD was observed to be non-linearly dependent on protein concentration, indicating that the active form of Mdl1p is a dimer. Surprisingly, the NBD did not reveal positive cooperativity with increasing ATP concentration. This is in contrast to the behavior of isolated MJ0796 NBD, which showed positive cooperativity with a Hill coefficient of 1.7 (29). However, in agreement with our data, other isolated NBDs like HisP and MalK displayed no cooperativity for ATP, while cooperativity in ATPase activity was determined in the fully assembled transporters HisQMP2 and MalFGK2 (25, 30). This different behavior of various ABC transporters shows that although dimerization of the NBDs is proposed for all, there are differences in the cooperativity of the NBDs, which in some cases might be caused by the TMDs.
The NBD of Mdl1p behaved as a monomer in gel filtration studies independent
of the presence of nucleotides as previously shown for other isolated NBDs
(25). For the first time, an
NBD of an ABC transporter was trapped as a dimer using either orthovanadate or
beryllium fluoride. ADP could not replace ATP in the trapping reaction,
demonstrating that ATP binding and hydrolysis are essential for dimerization.
Even in the absence of orthovanadate or BeFx, an ATP-dependent
dimer was obtained by mutation of the conserved glutamate downstream of the
Walker B motif of Mdl1p (E599Q). The formation of stable dimers upon ATP
binding was very recently described by gel filtration of mutant archaeal NBDs
(MJ0796-E171Q/MJ1267-E179Q)
(29). These mutants were
deficient in steady-state ATPase activity as assayed by standard ATPase assays
(Pi or ADP release) as were the equivalent mutants (E552Q/E1197Q or
E556Q/E1201Q) of human and mouse P-gp
(21,
28). The structures of some
ABC transporters have suggested that this conserved glutamate is the most
likely candidate to play the role of the catalytic carboxylate
(79,
11,
14,
17). During preparation of
this manuscript, this glutamate of human P-gp was suggested not to function in
the cleavage between and
phosphate, but to be part of the
switch region of ABC transporters possibly involved in the transmission of
interdomain signals from the substrate-binding site to NBDs
(21).
In this study, the nucleotide composition of the trapped states of Mdl1p
was analyzed in detail by incorporating [-32P]ATP or
[
-32P]ATP into the dimer. After gel filtration, the
nucleotide concentration was analyzed by
-counting and correlated to the
protein concentration. The nucleotide species were identified by TLC.
Radioactive nucleotides were exclusively associated with the dimers. This
observation demonstrated that ATP binding to the monomer is transient
resulting in either subsequent dimerization of the NBDs or dissociation of ATP
from the monomer. It was previously shown that nontrapped NBDs have very fast
koff rates for nucleotide binding
(23). Radioactive ADP in the
presence of various concentrations of ATP was not incorporated into the dimer.
Based on these results, ATP binding is regarded as essential and sufficient
for dimerization of the mutant. Interestingly, although AMP-PNP or ATP
S
binds with similar affinity to the E599Q mutant as ATP, none of these
analogues was able to induce ATP-dependent dimerization. This observation is
in line with results obtained on mutants from M. jannaschii
(29). Incubation of the
wild-type NBD of Mdl1p with an equimolar ratio of unlabeled MgATP and
MgATP
S under BeFx-trapping conditions did not result in any
incorporation of ATP
S in the dimer. This suggests that either the dimer
interface is disturbed in the presence of ATP
S, or both nucleotides
have to be hydrolyzed to obtain the BeFx-trapped intermediate.
Analysis of the dimer always showed two nucleotides bound per dimer. In case
of the BeFx-trapped NBD the nucleotide species was exclusively ADP
(with no Pi in the dimer), while in the E599Q mutant exclusively
two ATP molecules were incorporated. These data demonstrated that during the
ATPase cycle, both NBDs bind ATP. During gel filtration, which takes
1030 min, no nucleotides, but only the phosphate is released from the
formed dimer. Thus these results demonstrate that both nucleotides have to be
bound to both monomers before the stable dimer is formed, and that the two
nucleotides then remain associated during the entire hydrolysis cycle.
Surprisingly, incubation of the E599Q mutant with ATP in the presence of
Mg2+ at 30 °C resulted in slow hydrolysis of ATP,
indicating that the E599Q mutant still hydrolyzes ATP (but at a 3000-fold
reduced rate compared with the wild-type). This enabled us to explore the
nucleotide composition of the mutant E599Q dimer of Mdl1p under hydrolyzing
conditions. After prolonged incubation in the presence of limiting amounts of
ATP, an intermediate state, which contained one ATP and one ADP was formed.
This intermediate state could be isolated for a prolonged period of time. This
state was dependent on ATP limiting conditions, since in the presence of
excess of ATP a state with two ATPs was isolated (data not shown). Such an
asymmetric ATP/ADP state can only arise if the hydrolysis of the second ATP is
slower than hydrolysis of the first, and suggests that the ATP hydrolysis
occur by a sequential mechanism. Recently, mutants in the conserved glutamate
in human P-gp were shown to be able to hydrolyze one ATP molecule but unable
to initiate a second ATP hydrolysis
(20,
21,
28). The E599Q NBD of Mdl1p,
however, catalyzes multiple turnovers. This is probably caused by dimer
dissociation of the intermediate state with one ATP and one ADP, and rebinding
of ATP by the monomers, starting a new cycle in which again only one ATP is
hydrolyzed. Indeed under ATP limiting condition this dimer dissociates before
it hydrolyzed the second ATP (Fig.
6D, left panel). We tried to examine the
hydrolysis of the second ATP by following the fate of the nucleotides after
re-isolation of the dimer using gel filtration. Unfortunately, after dilution
by gel filtration, the association-dissociation kinetics of the equilibrium
between the monomeric and dimeric states and the rate of ATP hydrolysis were
of the same order (t
30 min), severely complicating our
analysis. To examine the coupling of ATP hydrolysis, Pi release,
and dimer dissociation in detail, either methods with a better time
resolution, or an E599Q mutant with a faster hydrolysis rate should be
used.
Interestingly, an asymmetric state with equimolar amounts of ATP and
ATPS was also observed after incubation of the E599Q mutant with ATP
and ATP
S into the dimer. This resulted in incorporation of one
ATP
S molecule per dimer (data not shown). The wild-type protein did not
incorporate ATP
S. This suggests that binding of ATP
S to the NBD
disturbs the dimer-dimer interface. The E599Q mutant seems to retain its
ability to form a dimer, if only one ATP
S is bound, while the wild-type
protein cannot accommodate the ATP
S in the dimer. The
-phosphate
contacts residues in both NBDs, thus stabilizing the dimer. Small changes in
the NBD around the
-phosphate thus strongly influence the ability of
the NBD to form dimers, and the wild-type protein seems more affected by this
than the E599Q mutant.
Published crystal structures of NBDs with a dimeric architecture showed
high symmetry of the two monomers and equal occupation of both
nucleotide-binding sites (14,
17) even in the full-length
structure of BtuCD (13).
However, based on vanadate trapping experiments combined with 8-azido-ATP
photo-labeling, for P-gp and homologues a model was proposed in which only one
NBD hydrolyzes ATP at a time, but in an alternating manner
(35,
36). Under hydrolysis
conditions, it was shown for P-gp as well as for MalFGK2 that
8-azido-ATP photolabeling of a BeFx- or vanadate-trapped state
resulted in approximately one [-32P]ADP bound per dimer
(23,
37,
38,
39). This stoichiometry of the
dimer is in contrast to our data. For all NBD dimers isolated we find two
nucleotides. Also in the BeFx-trapped state we obtained two
[
-32P]ADPs bound per dimer. This state contradicts the
alternating-site model. Our biochemical data is however supported by the
observed nucleotide occupancy in the crystal structures.
In summary, we have isolated three different intermediate states of the ATP hydrolysis cycle, containing either two ATPs, one ATP and one ADP, or two ADPs. Based on our experimental data we propose the processive clamp model, which is depicted in Fig. 7. As a first step in the ATPase cycle, ATP binds to the monomeric NBD. Since the ATP concentrations in the cell (for Mdl1p, in the mitochondrial matrix) are far above the Kd value determined for ATP binding, almost all of the NBDs would be in the ATP bound state under physiological conditions. Upon ATP binding, both NBDs associate to a dimer. This state is in a dynamic equilibrium between the association, dissociation as well as hydrolysis of the ATP. As a next step, one ATP is hydrolyzed. In the experiments with the E599Q mutant, hydrolysis of the second ATP could not be observed, because in the ATP/ADP bound state, the dimer is unstable and dissociates. In the slow hydrolysis cycle of the E599Q mutant, the dissociated monomers are reloaded with ATP and in a next cycle again one ATP is hydrolyzed. Hydrolysis in the wild-type NBD is however 3000-fold faster and here two ADPs can be trapped in the dimer. Based on the much faster kinetics of ATP hydrolysis and the trapping of two ADPs in the wild-type NBD, we suggest that both ATPs be hydrolyzed sequentially in one cycle, in a processive mode. Finally, after both ATPs are hydrolyzed, the dimer disassembles and both ADPs are released. How such a hydrolysis cycle is exactly coupled to transport of the substrate and conformational changes of the TMDs, and whether the TMDs superimpose a regulatory effect on the NBDs remains unknown and requires further investigations. Smith et al. (14) proposed a possible model for the coupling of ATP hydrolysis to substrate transport. In this model, the TMDs of the non-substrate bound transporter forced the NBDs to remain in their monomeric state. Binding of the substrate to the high affinity site in the TMDs changes the conformation of the TMDs and removes the restrain on the NBD. Only then, the ATP-loaded NBDs form a dimer. Formation of the dimer forces a change on the TMDs and displaces the substrate-binding site, which results in transport of the substrate across the membrane. Hydrolysis of the ATP and dissociation of the dimer subsequently resets the transporter. In such a model, which fits to both the crystal structures and our experimental data, the formation of the ATP-bound dimer would be the power stroke. This would also explain how substrate binding induces ATPase activity by transmission of conformational changes between TMDs and NBDs (36, 40, 41, 42). The mechanism of the coupling of ATP hydrolysis and substrate transport requires however further investigation.
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FOOTNOTES |
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Supported by a TALENT fellowship from the Netherlands Organization for
Scientific Research (NWO).
To whom correspondence should be addressed. Tel.: 49-69-798-29475; Fax:
49-69-798-29495; E-mail:
tampe{at}em.uni-frankfurt.de.
1 The abbreviations used are: ABC, ATP-binding cassette; AMP-PNP,
5'-adenylyl-,
-imidodiphosphate; ATP
S, adenosine
5'-O-(3-thio)triphosphate; ER, endoplasmic reticulum;
IC50, 50% inhibitory concentration; mAAA, matrix-oriented ATPases
associated with a variety of cellular activities; MALDI-TOF MS,
matrix-assisted laser desorption/ionization-time of flight mass spectrometry;
NBD, nucleotide-binding domain; PBS, phosphate-buffered saline; P-gp,
P-glycoprotein; TMD, transmembrane domain; TLC, thin layer chromatography;
TAP, transporter associated with antigen processing.
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ACKNOWLEDGMENTS |
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REFERENCES |
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