(Received for publication, June 28, 1995; and in revised form, July 25, 1995)
From the
Cystic fibrosis is caused by mutations in the cell membrane
protein called CFTR (cystic fibrosis transmembrane conductance
regulator) which functions as a regulated Cl channel.
Although it is known that CFTR contains two nucleotide domains, both of
which exhibit the capacity to bind ATP, it has not been demonstrated
directly whether one or both domains can function as an active ATPase.
To address this question, we have studied the first CFTR nucleotide
binding fold (NBF1) in fusion with the maltose-binding protein (MBP),
which both stabilizes NBF1 and enhances its solubility. Three different
ATPase assays conducted on MBP-NBF1 clearly demonstrate its capacity to
catalyze the hydrolysis of ATP. Significantly, the mutations K464H and
K464L in the Walker A consensus motif of NBF1 markedly impair its
catalytic capacity. MBP alone exhibits no ATPase activity and MBP-NBF1
fails to catalyze the release of phosphate from AMP or ADP. The V
of ATP hydrolysis (
30 nmol/min/mg of
protein) is significant and is markedly inhibited by azide and by the
ATP analogs
2`-(3`)-O-(2,4,6-trinitrophenyl)-adenosine-5`-triphosphate and
adenosine 5`-(
,
-imido)triphosphate. As inherited mutations
within NBF1 account for most cases of cystic fibrosis, results reported
here are fundamental to our understanding of the molecular basis of the
disease.
Cystic fibrosis is the most common autosomal recessive disease
in Caucasians affecting approximately 1 in 2000 people in the United
States and Canada(1, 2, 3) . The disease is
caused by mutations in the CFTR ()protein which impair its
normal function as a regulated phosphorylation-dependent Cl
channel in epithelial cells(2, 3) . CFTR, which
is comprised within a single polypeptide chain of 1480 amino acids, is
predicted to fold into five distinct domains, two nucleotide binding
folds (NBF1 and NBF2), a regulatory domain (R), and two transmembrane
spanning regions(4) . Although inherited mutations throughout
the CFTR protein are known to cause cystic fibrosis, there is a high
frequency of such mutations within or surrounding NBF1 and NBF2, with
those within NBF1, particularly
F508, being responsible for most
cases of the disease(1, 2, 3, 4) .
Therefore, in order to understand the molecular basis of the disease,
it is both necessary and fundamental to elucidate the functions of NBF1
and NBF2.
There is considerable evidence that both phosphorylation
of the CFTR protein, and its interaction with ATP, independent of
phosphorylation events, are required for optimal
functions(5, 6, 7, 8) . In the
latter case, the finding that nonhydrolyzable ATP analogs like AMP-PNP
fail to support CFTR function has led to the suggestion that ATP
hydrolysis may be required(7, 8) . Thus, unlike ATP,
AMP-PNP fails to open prephosphorylated Cl channels
in patch clamp experiments using Hela cells or 3T3 fibroblasts
expressing CFTR(7, 8) . Moreover, in single channel
analysis of artificial lipid planar bilayers reconstituted with human
CFTR, AMP-PNP, unlike ATP, failed to support channel
activity(9) .
Despite the above findings, it remains unresolved whether one or both of the two nucleotide binding folds of CFTR exhibit the capacity to hydrolyze ATP. In earlier reports from this laboratory(10, 11, 12) , we have demonstrated that peptide segments of both NBF1 and NBF2 containing the Walker A consensus motif (13) can bind TNP-ATP and that this analog can be displaced by ATP. However, no ATP hydrolytic capacity for either the NBF1 or NBF2 segment could be demonstrated (10, 11, 12) . Purified preparations of the complete NBF1, and of NBF1 in fusion with the maltose-binding protein (MBP-NBF1), have been obtained also(14, 15) . Similar to the peptide segments, NBF1 and MBP-NBF1 bind ATP and/or TNP-ATP(14, 15) . However, these preparations have not been examined in detail for their capacity to hydrolyze ATP.
Studies described in this report have focused on the MBP-NBF1 fusion protein which was originally overexpressed in Escherichia coli and purified in this laboratory(15) . Below, compelling evidence is provided that NBF1 within the MBP-NBF1 complex is an active ATPase.
The identity of the two base changes was confirmed by DNA sequencing(20) . Mutant proteins were overexpressed and purified as described above for wild type MBP-NBF1.
All studies reported here were carried out with the first
nucleotide binding fold (NBF1) of CFTR in fusion with the MBP. The
MBP-NBF1 fusion protein was prepared by an earlier method developed in
this laboratory (15) and modified as described under
``Experimental Procedures.'' Fig. 1summarizes results
of experiments in which this procedure was used to obtain highly
purified MBP-NBF1 for those studies described in this report. The
overexpression of the fusion protein in E. coli under control
of the tac promoter after induction with
isopropyl-1-thio--D-galactopyranoside, its elution from
an amylose column, and its electrophoretic profile on SDS-PAGE are
illustrated in Fig. 1, A-C, respectively. To
assure maximal purity of the product only a single peak fraction was
selected from the amylose column, which from SDS-PAGE is estimated to
be >95% pure. Purified MBP-NBF1 has the molecular mass expected of
60 kDa (42 kDa for MBP + 18 kDa for NBF1) and remains stable
for several months when stored as a 55% ammonium sulfate pellet at
-80 °C.
Figure 1:
Overexpression and purification of
MBP-NBF1. A, overexpression in E. coli. Cells
expressing MBP-NBF1 were grown as described under ``Experimental
Procedures.'' Where indicated cells were induced with 0.3
mM isopropyl-1-thio--D-galactopyranoside for 2 h
at 37 °C. After dissolving in SDS sample buffer, the whole cell
lysate (300- or 75-µl aliquots of 2
10
cells/ml
for uninduced and induced cells, respectively) was subjected to
SDS-PAGE. Molecular size markers are from top to bottom, phosphorylase b (97.4 kDa), bovine albumin
(66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin
inhibitor (21.5 kDa), and lysozyme (14.4 kDa). The arrow indicates the position of overexpressed MBP-NBF1. B,
purification of MBP-NBF1. The ammonium sulfate precipitate (see
``Experimental Procedures'') from 4 liters of cells was
dissolved in a buffer containing 20 mM Hepes, 50 mM NaCl, 1 mM EGTA, and 3 mM MgSO
, pH
7.4, and adsorbed onto a 2.5
5-cm amylose column. Elution was
carried out at 25 °C in the same buffer containing 20 mM maltose at a flow rate of 1 ml/min. Fractions (3 ml) were
collected and monitored at 595 nm using the Coomassie plus dye binding
assay. Fraction 4 in this and identical experiments was collected. C, SDS-PAGE of purified MBP-NBF1. Fraction 4 (3.4 µg) was
subjected to SDS-PAGE by the method of Laemmli(21) . Molecular
weight markers are identical to those in A.
To determine directly whether MBP-NBF1 has the
capacity to catalyze the hydrolysis of ATP, two completely different
assays, one based on the disappearance of the substrate ATP and the
other on the formation of the product ADP were used initially. The
assay based on the disappearance of ATP, which includes the
luciferin/luciferase reaction to monitor ATP levels, was employed
first. This is one of the most sensitive ATPase assays known, detecting
ATP concentrations in the nanomolar to micromolar range via
chemiluminescence. The assay is illustrated in Fig. 2A in a control experiment with a known ATPase, the mitochondrial
FF
-ATPase. Here, it can be seen that upon
addition of 3.3 µM ATP to a buffered system containing
only luciferin and luciferase a light flash (chemiluminesence response)
is detected which peaks and levels off to a near zero decay rate. Upon
addition of F
F
-ATPase, the chemiluminescence
signal rapidly decreases as ATP disappears upon its conversion to ADP
and P
. In Fig. 2B, it can be seen that when
this experiment is repeated with MBP-NBF1 a slower but significant rate
of disappearance of ATP is observed. Moreover, this rate is
ATP-dependent (Fig. 2B, inset) and remains linear at
the very low concentrations of ATP employed. Finally, in Fig. 2C it can be seen that MBP alone at concentrations
higher than MBP-NBF1 has no capacity to catalyze the hydrolysis of ATP,
implicating NBF1 as the catalytic unit. (It should be noted that MBP
has no nucleotide binding domains and neither binds nor hydrolyzes ATP (24) .)
Figure 2:
ATP hydrolytic activity of MBP-NBF1
monitored by following the disappearance of ATP in the
luciferin-luciferase chemiluminescence assay. After addition of 3.3
µM ATP, ATPase activity was monitored as a decrease in the
chemiluminescence signal following subsequent addition of A.
FF
-ATPase (3 µg); B, MBP-NBF1 (435
µg); and C, MBP (580 µg). In the inset in B the ATP hydrolytic rate is plotted versus ATP
concentration. Details of the assay procedure are summarized under
``Experimental Procedures.'' All experiments were repeated at
least twice and similar results were
obtained.
Although the chemiluminescence assay clearly
demonstrates that MBP-NBF1 and not MBP alone exhibits the capacity to
hydrolyze ATP, the assay is limited in the amount of ATP that can be
added. Thus, V values must be extrapolated
rather than determined directly. For this reason, the second assay
noted above, which monitors the formation of ADP coupled to the
pyruvate kinase and lactic dehydrogenase reactions, was employed. In
this coupled system, the ADP formed in the ATPase reaction is monitored
by the decrease in absorption of NADH when pyruvate is converted to
lactate. Fig. 3A shows that when increasing amounts of
MBP-NBF1 are added to the assay the rate of disappearance of NADH
increases. Fig. 3B shows that this increased rate of
hydrolysis is a linear function of the amount of MBP-NBF1 added
assuring that the increased rate is not due to a non-protein
contaminant. In data not presented, MBP alone had no capacity to
catalyze ATP hydrolysis as indicated above for the luciferin-luciferase
based assay. Results presented in Fig. 3C show that a
typical Michaelis-Menten plot is obtained when ATPase activity is
plotted versus ATP concentration resulting in K
and V
values, respectively, of 0.11 mM and
30 nmol/min/mg of protein (first order rate constant
= 0.5 min
/NBF1).
Figure 3:
ATP hydrolytic activity of MBP-NBF1
monitored by following the formation of ADP in a ``coupled''
spectrophotometric assay. ATPase activity was assayed by coupling the
release of ADP to the pyruvate kinase, lactic dehydrogenase reactions,
and measuring the decrease in absorbance at 340 nm exactly as described
under ``Experimental Procedures.'' A, chart tracings
showing the absorbance change at 340 nm under conditions where the
following amounts of MBP-NBF1 were added to the assay. , none;
, 0.24 mg;
, 0.48 mg;
, 0.75 mg;
, 0.96 mg;
, 1.44 mg. B, initial rates in A plotted versus mg of MBP-NBF1. C, plot of ATPase specific
activity of MBP-NBF1 versus ATP
concentration.
Results from the two
different assay procedures described above demonstrate that the highly
purified MBP-NBF1 fraction exhibits the capacity to catalyze ATP
hydrolysis. However, they do not rule out the possibility that a
contaminating protein, either another cell ATPase or a nonspecific
phosphatase, is responsible for the results obtained. For this reason
two different experiments were performed. In the first, ATP hydrolysis
was monitored by a third assay in which phosphate release is measured.
Results presented in Fig. 4A show that in the presence
of MBP-NBF1, phosphate is released from ATP as expected, but no
phosphate is released from AMP, and in data not presented from ADP;
phosphate release from AMP or ADP would be expected if a nonspecific
phosphatase were contaminating the preparation. Even more convincing
are results presented in Fig. 4, C and D,
where it is seen that the mutations, K464H and K464L, in the Walker A
nucleotide binding motif (GXGKT) of NBF1 reduce
the ATP hydrolytic capacity of purified mutant MBP-NBF1 proteins by
over 80%. Taken together, the two different types of experiments
described here leave little doubt that it is the NBF1 portion of
MBP-NBF1 that is responsible for its ATP hydrolytic capacity.
Figure 4:
Evidence that the ATP hydrolytic capacity
of MBP-NBF1 is not the result of a contaminating phosphatase and is a
property of NBF1. A, relative capacity of MBP-NBF1 to
hydrolyze the release of P from ATP and AMP. Phosphate
release was monitored colorimetrically exactly as described under
``Experimental Procedures'' in an assay system containing 5
mM ATP or 5 mM AMP, 5 mM MgCl
,
and 220 µg of MBP-NBF1. (In data not presented, ADP when
substituted for ATP, resulted in no phosphate release.) All assays were
carried out in duplicate. B, comparison of the purity of wild
type and mutant MBP-NBF1 proteins (K464H and K464L). SDS-PAGE by the
method of Laemmli (21) was carried out on 4 µg of wild-type
MBP-NBF1 and on 5 µg of the indicated mutant proteins. C and D, the effect of the mutations K464H and K464L within
NBF1 on the ATP hydrolytic activity of MBP-NBF1. ATP hydrolytic
activity was assayed using the coupled spectrophotometric method
exactly as described under ``Experimental Procedures.'' In C 550 µg of wild type and 670 µg of mutant protein
were included in the assay, whereas in D 270 µg of wild
type and 170 µg of mutant protein were included. (Note: specific
activity is plotted so that differences in protein concentrations are
normalized.) All experiments were repeated, and similar results were
obtained.
Finally, results presented in Fig. 5summarize those agents
which were found to inhibit the ATPase activity of MBP-NBF1 in the
coupled spectrophotometric assay (see ``Experimental
Procedures''). Here it is seen that, as in the case of the F moiety of F
F
-ATPase, the ATPase activity
of MBP-NBF1 is inhibited by the ATP analogs TNP-ATP (25) and
AMP-PNP (26) as well as by sodium azide. However, in contrast
to mitochondrial F
, where sulfite activates ATP hydrolysis
at least 2-fold(28) , the ATPase activity of MBP-NBF1 is
unaffected. In results not presented here, AMP-PNP was shown to be a
more effective inhibitor in the P
release assay.
Figure 5: The effect of known ATPase modulators on the ATPase activity of MBP-NBF1. ATPase activity was assayed by coupling the release of ADP to the pyruvate kinase, lactic dehydrogenase reactions, and measuring the decrease in absorbance at 340 nm exactly as described under ``Experimental Procedures.'' In all cases controls were run using ADP instead of MBP-NBF1 and ATP to assure that the modulators tested had no effect on the coupled enzymes. The indicated concentrations of modulators are shown. MBP-NBF1 (1.3 mg) was present in all assays, and all assays were carried out in duplicate. Experiments were repeated at least once with all modulators.
This is the first report to provide direct evidence in
vitro that the first nucleotide binding fold (NBF1) of the CFTR
protein can function as an active ATPase. Assays conducted on the
MBP-NBF1 fusion protein monitored either disappearance of the substrate
ATP (Fig. 2B) or appearance of the products ADP (Fig. 3) and P (Fig. 4A) leaving no
doubt that the purified protein catalyzes the hydrolysis of ATP. The
additional experimental results demonstrating that MBP alone has no
catalytic capacity (Fig. 2C) and that mutations (K464H
and K464L) within the Walker nucleotide binding motif
GX
GKT markedly inhibit ATPase activity (Fig. 4, C and D) localize the catalytic site
to NBF1. These results do not exclude the possibility that within
intact CFTR the NBF1 domain may require stabilizing interactions with
another domain. Thus, it should be noted that in the F
moiety of the F
F
-ATPase, the
subunit, although containing all residues essential for catalysis, is
itself not catalytic(29, 30) . Rather, interaction
with the noncatalytic
subunit is required to elicit ATP
hydrolysis(31) .
Relative to other proteins that catalyze
the hydrolysis of ATP, the V rate of MBP-NBF1 of
30 nmol/min/mg is slightly higher than that observed for
chaperones (32) but considerably lower than that observed for
the CFTR related MDR-protein (multidrug resistance
protein)(33, 34) . The much lower rate characteristic
of NBF1 of CFTR may reflect a role in using ATP hydrolysis to only open
Cl
channels, as opposed to transporting a drug, as in
the case of MDR. However, it remains possible that the activity
observed here for CFTR NBF1 may be a minimal value and that in vivo hitherto unknown substrates are transported by CFTR, in which case
a higher ATP hydrolytic rate may be required. Significantly, it has
been reported recently that in addition to Cl
, CFTR
can also translocate ATP(27, 35) .