Biochemical Evidence for Interaction between the Two
Nucleotide Binding Domains of ArsA
INSIGHTS FROM MUTANTS AND ATP ANALOGS*
Hongwei
Jia and
Parjit
Kaur
From the Department of Biology, Georgia State University, Atlanta,
Georgia 30303
Received for publication, November 20, 2002
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ABSTRACT |
ArsA, the peripheral membrane component of the
anion-translocating ATPase ArsAB, consists of two nucleotide binding
domains (A1 and A2), which are connected by a linker sequence.
Previous studies on ArsA have focused on the function of each
nucleotide binding domain and the role of the linker, whereas the
present study looks at the interactions between the binding domains and their interactions with the linker. It has previously been shown that
the A1 domain of ArsA carries out unisite catalysis in the absence of
antimonite, while A2 is recruited in multisite catalysis by antimonite
in the presence of a functional A1 domain. Multisite catalysis thus
seems to result from an interaction between A1 and A2 brought about by
antimonite. In the present study, we provide direct biochemical
evidence for interaction between the two nucleotide binding domains and
show that the linker region acts as a transducer of the conformational
changes between them. We find that nucleotide binding to the A2 domain
results in a significant, detectable change in the conformation of the
A1 domain. Two ATP analogs, FSBA and ATP
S, used in this study, were
both found to bind preferentially to the A2 domain, and their binding
resulted in changing the otherwise compact A1 domain into an open
conformation. Point mutations in the A2 domain and the linker region
also produced a similar effect on the conformation of A1, thus
suggesting that events at A2 are relayed to A1 via the linker. We
propose that nucleotide binding to A2 produces a two-tiered
conformational change. The significance of these changes in the
mechanism of ArsA is discussed.
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INTRODUCTION |
ArsA is the peripheral membrane component of the
anion-translocating ATPase ArsAB. It is an allosteric enzyme whose
ATPase activity is stimulated by the substrates of the pump, antimonite and arsenite (1). ArsA consists of two homologous halves, A1 (residues
1-281) and A2 (residues 321-583), each half consisting of a consensus
nucleotide binding domain
(NBD)1 characterized by the
presence of a Walker A sequence (2). The two halves of ArsA are
connected by a linker sequence (residues 282-320), which plays a
crucial role in the function of ArsA (3). Site-directed mutations
created in specific residues in each NBD suggest that both sites are
required for function of the protein (4, 5). We have proposed earlier
that the two NBDs in ArsA have different conformations and different
affinities for ATP (6). In a previous publication from this laboratory,
we proposed that ATP binds only to the A1 site in the absence of
antimonite, whereas it binds to both A1 and A2 in its presence. Thus,
the function of the allosteric ligand may be to act as a switch that allows ATP binding to A2, resulting in A1-A2 interaction and catalytic co-operativity (6). Even though sequential ATP binding to A1 and A2 and
the role of antimonite in allowing ATP binding to A2 has not been
unequivocally verified, we have shown that antimonite is required for
recruiting A2 into catalysis (7). We showed that the A1 site carries
out catalysis in the absence of antimonite, whereas A2 participates in
catalysis in the presence of antimonite and a functional A1 site (7).
Catalysis by A1 in the absence of antimonite has been termed "unisite
catalysis" and catalysis involving both A1 and A2 in the presence of
antimonite "multisite catalysis" (7). Studies carried out with
point mutants in the A1 and the A2 domains showed that proteins
containing mutations in A2 still carry out unisite catalysis in A1, but
no multisite catalysis is seen (7). However, proteins containing
mutations in A1 show neither unisite nor multisite catalysis (7). These results showed that not only is the A1 site first in the sequence of
events but for participation of A2 in catalysis, a functional A1
domain, in addition to antimonite, is required (7). In a recent study
(8), Rosen and co-workers studied intrinsic tryptophan fluorescence of a residue near A2 to understand the role of A2 NBD.
Their data are consistent with the proposed unisite and multisite catalytic mechanism (7) of ArsA and demonstrate independently that A2
is catalytic only when antimonite is present and when the A1 site is
functional (8).
The studies discussed above point to a sequence in the catalytic
mechanism of ArsA, and at the same time suggest interaction between the
A1 and the A2 domains, which may be responsible for catalytic
co-operativity. The present work provides direct biochemical evidence
for interaction between the domains. We find that events at the A2
domain result in a significant conformational change in the A1 domain,
thus providing strong evidence for interaction. Our results also
indicate that the linker region acts as the mediator of the
conformational changes between A1 and A2 and that events from A2 are
transduced to A1 via the linker.
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MATERIALS AND METHODS |
Purification of ArsA
His-tagged wild type and mutant ArsA proteins were purified by
Ni-nitrilotriacetic acid chromatography according to the procedure described earlier (6).
Trypsin Sensitivity Assay
ArsA (10 µM) in a 100-µl reaction volume
containing MOPS-KOH, pH 7.5 was incubated with the indicated substrates
for 10 min at 37 °C. The samples were then subjected to different
concentrations of trypsin, as indicated, for 60 min at 25 °C.
Proteolysis was terminated by addition of 2-fold excess soybean trypsin
inhibitor. The samples were analyzed by SDS-PAGE using 12%
polyacrylamide gel, followed by Coomassie Blue staining or Western blot
analysis using anti-N18 (9) or anti-C30 (6) antibodies.
Inhibition of the ATPase Activity of ArsA by ATP
S
To determine the inhibitory effect of ATP
S on the ATPase
activity of ArsA, either the coupled assay (5) or the
32Pi release assay (7) was employed, as
described below.
Coupled Assay--
ArsA (15 µg) was incubated with the
reaction components of the coupled assay and the indicated
concentrations of ATP
S in a 1-ml cuvette. Antimonite (0.5 mM) and ATP (1 mM) were added, and the samples
were preincubated at 37 °C for 10 min. The reactions were started by
the addition of magnesium and decrease in absorbance of NADH was
monitored at 340 nm. A control experiment showed that ATP
S has no
effect on the components of the coupled assay system itself. To
determine the protective effect of ATP on ATP
S inhibition, ArsA was
incubated with different concentrations of ATP, antimonite (0.5 mM), and a fixed concentration of ATP
S in the coupled
assay system for 10 min. The reaction was started by the addition of magnesium.
32Pi Release Assay--
ArsA (1 µg)
was preincubated with [
-32P]ATP (2.5 µCi) and
different concentrations of ATP
S in a 40-µl reaction volume in 50 mM MOPS-KOH, pH 7.5 for 10 min at 37 °C. Antimonite was
added to the preincubation mixture where indicated. The reaction was started by the addition of magnesium, and the samples were withdrawn at
different times, followed by TLC.
Labeling of ArsA with
-35S-ATP
Purified ArsA (10 µM) was incubated with
-35S-ATP (10 µCi) in 100 µl of buffer A (50 mM Tris-Cl, pH 7.5, 10% glycerol, 1 mM EDTA)
for 10 min at room temperature. The samples were placed in a
microtitration plate and subjected to UV cross-linking by placing a
hand-held UV lamp (254 nm) directly above the microtitration plate for
30 min on ice. The samples were precipitated with 10% trichloroacetic
acid on ice for 30 min, followed by centrifugation and resuspension of
the pellet in 100 µl of the buffer A. The samples were analyzed by
SDS-PAGE on a 10% polyacrylamide gel, followed by autoradiography.
Identification of the Site of Labeling with
-35S-ATP in ArsA
ArsA, UV cross-linked with
-35S-ATP in buffer A,
as described above, was directly mixed with trypsin at a
trypsin/protein ratio of 1:1000. The samples were incubated at 25 °C
for 60 min, followed by addition of 2-fold excess trypsin inhibitor.
The samples were then analyzed by SDS-PAGE on a 12% gel. The dried gel
was subjected to fluorography and autoradiographed.
Amino Acid Sequencing of the Peptide Fragments
Trypsin-digested fragments were transferred onto a
polyvinylidene difluoride membrane. The bands were excised and
subjected to N-terminal sequencing using a Beckman model LF3000 solid
phase amino acid sequencer in the core facility of the Department of Biology at the Georgia State University.
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RESULTS |
Previous studies have shown that Arg-290 in the linker region
(residues 282- 320) of ArsA is a readily accessible trypsin site (6).
At a ratio of trypsin to ArsA of 1:1000, cleavage at Arg-290 divides
the protein into two halves, a 32-kDa N-terminal fragment (residues
1-290) and a 27-kDa C-terminal fragment (starting at residue 290). The
N-terminal 32-kDa fragment is very compact and is resistant to further
cleavage by trypsin, whereas the 27-kDa fragment is cleaved further
into smaller fragments (6). This digestion pattern is seen in the
control sample (no substrate) as well as in the presence of ATP.
Interestingly, however, in the presence of ATP and antimonite, ArsA
acquires a trypsin-resistant conformation, which has been designated AS
as compared with the A conformation obtained in the presence of ATP
alone (6). Most ArsA in the AS conformation is found in the 63-kDa
species, indicating that Arg-290 is now buried in the structure and is
inaccessible to trypsin. In this study, we looked at the ability of the
point mutants in the nucleotide binding sites of ArsA to acquire the AS
conformation. Two point mutants were chosen, G20S with a
mutation in the A1 NBS (4) and K340E with a mutation in the A2 NBS (5). The data in Fig. 1 suggest that, unlike
in the wild type ArsA (lanes 1-3), neither G20S nor K340E
can acquire the AS conformation in the presence of ATP and antimonite.
The digestion pattern of K340E is exactly the same under three
different conditions, which include the control (no substrate,
lane 7), A (ATP, lane 8), or the AS (ATP and
antimonite, lane 9). The N-terminal 32-kDa fragment, which
results from cleavage at Arg-290, is the predominant fragment in all
situations (Fig. 1, lanes 7-9). The AS conformation is also
completely absent in G20S (Fig. 1, lanes 4-6).

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Fig. 1.
AS conformation in wild type and mutant ArsA
proteins. Purified wild type or mutant (A1 mutant G20S and A2
mutant K340E) ArsA proteins (10 µM) were incubated with 5 mM ATP and 0.5 mM antimonite for 10 min at
37 °C and subjected to trypsin proteolysis at a trypsin/protein
ratio of 1:1000 for 60 min, as described under "Materials and
Methods." The samples were analyzed by 12% SDS-PAGE, followed by
staining with Coomassie Blue. Lanes 1-3, wild type ArsA;
lanes 4-6, G20S; lanes 7-9, K340E; lanes
1, 4, and 7, no substrate; lanes 2, 5, and
8, 5 mM ATP; lanes 3, 6, and
9, 5 mM ATP and 0.5 mM
antimonite.
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Since the AS conformation of ArsA is resistant to trypsin at a
concentration of 1:1000, trypsin concentrations of 1:100 and 1:50 were
employed to analyze the AS conformation further. The data in Fig.
2A show that Arg-290 in AS is
relatively inaccessible even at high trypsin concentrations, however a
new 40-kDa fragment is produced exclusively in the AS sample
(lanes 3 and 5) and not in the A sample
(lanes 2 and 4). Western blot analysis (Fig.
2B) showed that this band cross-reacts with both anti-N18
(residues 1- 176) (lane 2) and anti-C30 (residues 320-583)
(lane 4) antibodies, thus suggesting that it originates
within the A1 domain and extends into the A2 domain. This fragment was
designated the NC40 fragment. An N-terminal amino acid sequence
analysis of this fragment showed that it originates at residue Arg88 in
the A1 domain and it bears the amino acid sequence
89IVDPIKGVL (Table I). Since
this site is inaccessible to trypsin in the control (no substrate) or
in the A (with ATP) conformation, it suggests that an alternate trypsin
site within the A1 domain becomes available in the AS conformation,
albeit only at high concentrations of trypsin. (See Fig.
3 for a linear depiction of ArsA.)

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Fig. 2.
Trypsin analysis of the AS conformation of
ArsA. Wild type ArsA (10 µM) incubated with 5 mM ATP and 0.5 mM antimonite for 10 min at
37 °C was subjected to trypsin digestion at a trypsin/protein ratio
of 1:100 or 1:50 for 60 min. The samples were analyzed by 12%
SDS-PAGE. A, Coomassie Blue staining. Lane 1,
undigested control; lanes 2 and 4, 5 mM ATP; lanes 3 and 5, 5 mM ATP and 0.5 mM antimonite. B,
samples digested with 1:100 trypsin were analyzed by Western blot using
anti-N18 and anti-C30 serum. Lanes 1 and 2, anti
N-18; lanes 3 and 4, anti-C30; lanes 1 and 3, 5 mM ATP; lanes 2 and
4, 5 mM ATP and 0.5 mM
antimonite.
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Table I
N-terminal amino acid sequence of various peptides generated by trypsin
or cyanogen bromide cleavage
This table includes different peptides of ArsA that either contain the
binding sites (C18, C27, C14) for the ATP analogs or are the result of
alternate trypsin cleavage in A1 due to ATP/ATP analog binding in A2.
Abbreviations and notations: AS, ArsA incubated with ATP and
antimonite; FSBA, ArsA incubated with FSBA; ATP S, ArsA incubated
with ATP S; D303G mutation, ArsA containing D303G linker mutation. An
X in the amino acid sequence indicates a residue that could
not be definitively identified.
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Fig. 3.
A linear depiction of the wild type
ArsA. The two halves of ArsA, A1 and A2, and the intervening
linker region (hatched lines) are shown. Trypsin cleavage
sites, Arg-88 in A1 and the Arg-290 in the linker, are marked. Various
peptides of ArsA resulting from cleavage at Arg-290 (N32 and C27) or
the alternate site Arg-88 (N25 and NC40) are shown below the linear map
of ArsA. The binding sites for the ATP analogs, FSBA (355-482,
stippled) and -35S-ATP (291-442,
vertical lines), are also marked.
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To determine if availability of the alternate trypsin site (Arg-88) in
A1 depends on nucleotide binding to A2, analogs of ATP, including FSBA
and ATP
S, were used. FSBA inhibits the ATPase activity of ArsA in a
specific manner (10). It was chosen for this study, in particular,
because it binds preferentially to the A2 domain of ArsA, and its
binding results in protection of the C27 fragment from proteolysis at a
trypsin concentration of 1:1000 (6). In the present work, we find that
binding of FSBA to ArsA results not only in the protection of the C27
fragment but it also produces another fragment of about 25 kDa in size, which is present exclusively in the FSBA-treated ArsA samples (Fig.
4A, lane 3) and not present in
the control (lane 1) or the ATP (lane 2) sample.
To determine the origin of this fragment, Western blot analysis was
carried out. Data in Fig. 4B show that the 25-kDa band
cross-reacts with the anti-N18 antibody (lane 3) but not
with the anti-C30 antibody (lane 6), thus implying that this
fragment is the result of further breakdown of the otherwise compact
N32 fragment (residues 1-290). This fragment is designated N25.
N-terminal amino acid sequence analysis showed that this fragment
contains the sequence 89IVDPIKGVLP, thus this fragment also
results from cleavage at Arg-88 in A1 (Table I). Based on its size of
25 kDa, this fragment is expected to lie between Arg-88 and Arg-290.
Effect of FSBA binding to the point mutants, G20S and K340E, was also
studied. The data in Fig. 4A show that the N25 fragment is
produced when G20S is treated with FSBA (lane 6).
Interestingly, however, trypsin proteolysis of K340E results in the N25
fragment irrespective of the presence or absence of FSBA (lanes
7-9).

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Fig. 4.
Effect of FSBA binding on the conformation of
the A1 domain of ArsA. A, wild type or mutant (G20S and
K340E) ArsA proteins incubated with 5 mM ATP or 1 mM FSBA for 10 min at 37 °C were subjected to trypsin
proteolysis at 1:1000 for 60 min. The samples were analyzed by 12%
SDS-PAGE and stained with Coomassie Blue. Lanes 1-3, wild
type ArsA; lanes 4-6, G20S; lanes 7-9, K340E.
Lanes 1, 4, and 7, no substrate;
lanes 2, 5, and 8, 5 mM
ATP; lanes 3, 6, and 9, 1 mM FSBA. B, wild type ArsA samples from
A were analyzed by Western blotting using anti-N18 or
anti-C30 antibodies. Lanes 1-3, anti-N18; lanes
4-6, anti-C30; lanes 1 and 4, no substrate;
lanes 2 and 5, 5 mM ATP; lanes
3 and 6, 1 mM FSBA.
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Effect of binding of another ATP analog, ATP
S, on the function of
ArsA was also studied. Initial experiments focused on determining if
ATP
S inhibits the ATPase activity of ArsA and in localization of its
binding site in ArsA. To determine if ATP
S inhibits the ATPase
activity, ArsA was preincubated with the indicated concentrations of
ATP
S in the presence of antimonite and ATP, and its effect on the
ATPase activity was determined by the coupled assay, as described under
"Materials and Methods." The ATPase activity of ArsA was inhibited
in a dose-dependent manner (Fig.
5). Half-maximal inhibition was observed
at 20-40 µM ATP
S. ATP was found to protect against
the inhibitory effect of ATP
S. Complete protection from inhibition
was observed at 8 mM ATP (data not shown), suggesting that
ATP
S binds more tightly to one or both sites in ArsA as compared
with ATP.

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Fig. 5.
Dose-dependent inhibition of the
ATPase activity of ArsA by ATP S. 15 µg
of ArsA was incubated with different concentrations of ATP S in 1 ml
of the coupled ATPase assay reaction mixture in 50 mM
MOPS-KOH (pH 7.5), 0.25 mM EDTA. The reaction mixture also
contained 1 mM ATP and 0.5 mM antimonite. After
incubation for 10 min at 37 °C, the reactions were initiated by the
addition of MgCl2. A decrease in absorbance at 340 nm was
monitored. The percentage ATPase activity was plotted as a function of
the ATP S concentration. The solid line represents a
non-liner regression of the data and IC50 of about 20 µM.
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To understand the mechanism of ATP
S inhibition, its effect on the
kinetic parameters was determined (Fig.
6). ArsA was preincubated in the presence
of antimonite, ATP, and different concentrations of ATP
S. In the
control reaction carried out in the absence of ATP
S, a
Km of 131 µM and a
Vmax of 966 nmol/min/mg ArsA was observed. These
values are consistent with the values reported earlier (1, 5). In the
presence of increasing concentrations of ATP
S, an increase in
Km was observed, whereas Vmax was found to remain unchanged (Fig. 6). These data show that in the
presence of ATP
S, affinity of ArsA for ATP is significantly decreased, suggesting that ATP
S is a competitive inhibitor that binds with a higher affinity than ATP.

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Fig. 6.
Kinetic analysis of ATP hydrolysis by ArsA in
the presence of ATP S. The experimental
conditions were same as in Fig. 5. Effect of different concentrations
(squares, 0 µM; triangles, 10 µM; inverted triangles, 20 µM;
diamonds, 40 µM) of ATP S on hydrolysis of a
range of ATP concentrations by ArsA was determined. The data are
presented in the form of a double-reciprocal plot. A
Vmax = 946.9 ± 114.3 nmol/min/mg and
Km = 131.2 µM were obtained in the
absence of the inhibitor. The Ki values obtained in
the presence of ATP S were 1.48 mM (10 µM
ATP S), 4.38 mM (20 µM ATP S), and 4.54 mM (40 µM ATP S).
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ArsA has previously been shown to exhibit unisite catalysis from the A1
site in the absence of antimonite and multisite catalysis involving A1
and A2 in the presence of antimonite (7, 8). The effect of different
concentrations of ATP
S on the initial rate of unisite and multisite
catalysis was determined by the 32Pi release
assay. Preincubation with ATP
S was carried out in the presence of
ATP and either the presence (multisite conditions) or the absence of
antimonite (unisite conditions). Data in Fig. 7 show that the unisite activity of ArsA
(squares) is unaffected even at concentration as high as 100 µM ATP
S, thus suggesting that ATP
S either does not
bind to the A1 site or it does not inhibit the unisite activity of A1.
However, a significant decrease in the initial rate of activity in the
presence of antimonite was seen (triangles), which may
suggest binding of ATP
S primarily to the A2 domain of ArsA.

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Fig. 7.
Effect of ATP S on
unisite and multisite catalytic activity of ArsA.
32Pi release assay was used to determine the
effect of ATP S on the initial rate of hydrolysis by ArsA either in
the absence (squares) or presence (triangles) of
0.5 mM antimonite. The samples were withdrawn at different
time points (20, 40, 60, 80, and 100 s in the presence of
antimonite and 1, 2, 3, 4, and 5 min in the absence of antimonite) and
analyzed by TLC.
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To identify the ATP
S binding site, ArsA was mixed with
-35S-ATP and exposed to UV light as described under
"Materials and Methods." Wild type ArsA was seen to form a
UV-activated adduct with
-35S-ATP (Fig.
8, lane 1). The addition of
antimonite or magnesium was not found to increase the efficiency of
labeling (data not shown). UV-induced photolabeling in the presence of
-35S-ATP was also studied in the A1 (G20S) or the A2
(K340E) mutants. The data in Fig. 8 show that the A1 mutant, G20S,
forms adduct with
-35S-ATP (lane 2), while no
labeling is seen in the A2 mutant, K340E (lane 3). These
results thus suggest that labeling with
-35S-ATP most
likely occurs to the A2 domain.

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Fig. 8.
Characterization of the UV-activated adduct
between ArsA and -35S-ATP.
UV-activated adduct formation between wild type or mutant ArsA proteins
(10 µM) and 35-S-ATP was performed as
described under "Materials and Methods." The samples were analyzed
by 10% SDS-PAGE, followed by autoradiography. Lane 1, wild
type ArsA; lane 2, G20S; lane 3, K340E.
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To localize the binding site of
-35S-ATP in ArsA,
partial trypsin proteolysis of the labeled proteins was carried out.
Under the trypsin conditions employed (described under "Materials and Methods"), ArsA is cleaved at Arg-290 into an N-terminal 32-kDa and a
C-terminal 27-kDa fragment (Fig.
9A, lanes 3 and
4). We find that in a buffer containing 10% glycerol (as
used in this experiment), the C27 fragment is stable and is not
digested further, thus allowing us to determine the relative labeling
of N32 versus C27 with
-35S-ATP. The
autoradiogram in Fig. 9B shows that the C-terminal 27-kDa
fragment is the predominant labeling site in wild type ArsA and in G20S
(lanes 3 and 4). A smaller fragment of about 18 kDa is also labeled (lanes 3 and 4). The
N-terminal amino acid sequence of the 18-kDa fragment was determined
and it corresponds to 291LXSXQPVA, indicating that it is
the breakdown product of C27 (Table I and Fig. 3).

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Fig. 9.
Localization of the UV-activated adduct
between ArsA and -35S-ATP.
Wild type or G20S ArsA was UV cross-linked with -35S-ATP
as in Fig. 8. Labeled proteins were subjected to trypsin proteolysis as
described under "Materials and Methods." The ratio of
trypsin/protein was 1:1000, and samples were incubated at 25 °C for
60 min. The samples were analyzed by 12% SDS-PAGE. A,
Coomassie Blue staining. B, gel in A was dried
and subjected to autoradiography. Lanes 1 and 3,
wild type ArsA; lanes 2 and 4, G20S; lanes
1 and 2, undigested samples; lanes 3 and
4, samples digested with trypsin.
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The data from the experiments outlined above suggest that
ATP
S, like FSBA, binds to the A2 domain of ArsA. If so, does binding of ATP
S to A2 also produce a conformational change in A1? Trypsin analysis showed that binding of ATP
S, in the presence of magnesium, releases a 40-kDa fragment (Fig. 10,
lane 5). N-terminal sequence analysis of the 40-kDa fragment
showed that it also results from cleavage at Arg-88 and contains the
sequence 89IVDPIKGVL (Table I). This fragment is thus
similar to the NC40 fragment produced by trypsin cleavage of ArsA in
the AS conformation and it, once again, suggests a conformational
change in A1 upon binding of nucleotide to A2.

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Fig. 10.
Effect of ATP S
binding on trypsin proteolysis of ArsA. Wild type ArsA was
incubated with different substrates, as indicated, for 10 min at
37 °C and subjected to trypsin proteolysis at 1:1000 for 60 min.
Samples were analyzed by 12% SDS-PAGE, followed by staining with
Coomassie Blue. Lane 1, 5 mM ATP; lane
2, 5 mM ATP and 0.5 mM antimonite;
lane 3, 1 mM ATP S; lane 4, 1 mM ATP S and 0.5 mM antimonite; lane
5, 1 mM ATP S and 5 mM
MgCl2.
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DISCUSSION |
This article describes the long range conformational changes that
occur in the A1 domain of ArsA on nucleotide binding to the A2 domain
and elucidates the role of the linker in transducing these changes. It
is previously known that ArsA, in the presence of ATP and antimonite,
acquires a conformation strikingly different from the conformation
produced in the presence of either by itself (6). We have proposed
earlier that the A conformation of ArsA results from ATP binding to the
A1 site, whereas the AS conformation results from binding of ATP to A1
and A2 in the presence of antimonite, followed by interaction between
the domains (6). The significance of the AS conformation of ArsA is
evident from the fact that point mutants in either NBD of ArsA do not
produce the AS conformation (Fig. 1). This suggests that AS
conformation is indeed the result of interaction between two functional
NBDs with ATP bound to one or both. AS conformation is the
trypsin-resistant conformation so that Arg-290 in the linker is not
accessible to trypsin. However, we find that at a higher concentration
of trypsin, an alternate site, Arg-88, becomes accessible in AS.
Cleavage at Arg-88 results in the NC40 fragment. These data imply a
conformational change in A1 resulting from its interaction with A2 in
the presence of antimonite. Do these data also indicate that ATP
binding to A2 in the presence of antimonite results in a conformational
change in A1? To answer that question, further experimental evidence was collected.
Since FSBA, an ATP analog, binds preferentially to the A2 domain of
ArsA (6), we asked if binding of FSBA to the A2 domain produces a
conformational change in A1. In this study, we find that FSBA binding
to A2 protects C27 from further cleavage by trypsin (as shown before in
Ref. 6), and, at the same time, it produces a conformational change in
the A1 domain making it accessible to trypsin (Fig. 4). This results in
a new fragment N25, which originates from Arg-88 in A1 (Figs. 3 and 4).
Thus FSBA binding studies demonstrate very clearly that nucleotide binding to A2 results in a conformational change in A1. Interestingly, our results also show that a mutation in the A2 domain (e.g.
K340E) results in the N25 fragment both in the presence or absence of FSBA, suggesting that the A1 domain in K340E is locked in an open conformation, thus providing further evidence that the conformational change in A1 is induced by events at A2.
Furthermore, we find that binding of another ATP analog, ATP
S, to
the A2 domain of ArsA also results in a conformational change in A1. A
40-kDa fragment, originating at Arg-88, is produced in the presence of
ATP
S and magnesium (Fig. 10). This 40-kDa band is similar to the
NC40 fragment produced on trypsin cleavage of the AS conformation (Fig.
1). Thus, cleavage at Arg-88 on nucleotide binding to A2 results in
either the N25 (with FSBA) or the NC40 (with ATP or ATP
S) fragment
depending, most likely, on the binding characteristics of the
nucleotide in question (for binding sites, please see Fig. 3).
The two NBDs of ArsA are connected by a linker sequence, which has been
shown to play a crucial role in the function of the protein (3).
Several mutations in the linker region were reported earlier (3). One
mutation, D303G, was found to be particularly interesting (3). In
addition to resulting in high arsenite sensitivity, this mutation was
shown to produce a conformational change in the A1 domain resulting in
a new fragment N25 (3). In light of the studies reported here, this
observation takes on a new significance. We decided to determine its
N-terminal amino acid sequence and found that it too results from
cleavage at Arg-88 (Table I). Thus a mutation in the linker (D303G) has the same effect on the conformation of A1 as a mutation in A2 (K340E),
or binding of an ATP analog to A2 (FSBA). It implies, as suggested
earlier (3), that interaction between A1 and A2 involves the linker.
That these three components (A1, A2, linker) interact closely is
further evidenced by the fact that a mutation in any of these three
components prevents the formation of the AS conformation (Fig. 1 and
Ref. 3). It suggests that A1 communicates with A2 via the linker, and
this flow of information is disrupted in the D303G mutation (no AS is
seen). Thus, the linker functions as a transducer of conformational
changes from A1 to A2 and vice versa. In summary, the point
mutants in the two binding sites and the linker allow us to look at the
conformational changes that would normally occur in the wild type ArsA.
Whereas wild type ArsA switches from A to AS on addition of
antimonite, the point mutants in any of the three domains do not make
this transition. The A2 mutation and the D303G linker mutation,
however, have the effect of locking the A1 domain in a particular
trypsin-sensitive conformation, irrespective of whether nucleotide is
bound to A2 or not. Thus there is evidence for two separate effects
resulting from events at A2: a long range conformational change in A1
(reflected in trypsin accessibility of Arg-88), and a movement of the
A1 and A2 domains (reflected in the AS conformation and inaccessibility of Arg-290). In the wild type ArsA the long range conformational change
in A1 is accompanied by the formation of the AS, whereas in the mutants
it is not.
There is no doubt that these conformational changes reflect interaction
between A1 and A2, most likely via the linker; however, this raises the
question: what is the significance of these changes in the mechanism of
catalysis by ArsA? The answer may be linked to the asymmetric nature of
the two sites in ArsA. We have proposed that the two sites in ArsA are
non-equivalent and that only the A1 NBD is competent to bind ATP in the
absence of antimonite, whereas ATP binding to A2 is "switched on"
in the presence of antimonite (6). Even though clear evidence for this
proposal is still lacking, this could very well be the basis for
sequential participation of A1 and A2 in catalysis (7). We have shown that A1 participates in catalysis first in the absence of the ligand
(unisite catalysis), whereas A2 comes into play later, in the presence
of the ligand and a functional A1 (multisite catalysis). This has
recently been verified in independent studies carried out by Zhou
et al. (8). Thus the conformational changes observed in this
study may play a role in the cycle of ATP binding and ADP release from
A1 and A2. The following sequence of steps might be envisioned: ATP
binding and hydrolysis occurs in A1 first in the absence of antimonite,
which produces a change in A2 so that A2 is now competent to bind ATP.
If antimonite is available, ATP binding to (and perhaps hydrolysis by)
A2 (in turn) produces a conformational change in A1, causing release of
tightly bound ADP from A1 and allowing binding of another molecule of
ATP to A1. Whether antimonite indeed acts as a switch for ATP binding to A2 could be answered by structural studies, however, crystals of
ArsA in the absence of antimonite have not been obtained (11). Nevertheless, the crystal structure studies have confirmed that the two
sites in ArsA are non-equivalent and that the exchange with nucleotides
occurs only at the A2 site (11). Further, these studies have also shown
that the nature of the nucleotide bound at A2 is reflected in the
conformation of the A1 domain (12). Simultaneous changes in the
conformation of the linker region of ArsA have also been reported (12).
Thus, our biochemical studies are, to a large extent, supported by the
crystal structure data.
Finally, a comparison of ArsA to CFTR and P-glycoprotein, two
other proteins that contain two NBDs each (13, 14) suggests that ArsA
is more similar to CFTR in its mode of function than it is to
P-glycoprotein. The two NBDs in CFTR and ArsA seem to be non-equivalent
and participate sequentially in the catalytic cycle; whereas in Pgp,
the two sites seem to be equivalent and even though they alternate in
catalysis, there is no sequence to the steps. Each cycle of hydrolysis
and transport in Pgp involves hydrolysis of only one ATP at either
site. However, in both CFTR and ArsA, the N-terminal NBD is the first
site to come into play; for one complete cycle, both NBDs seem to be
involved. For example, in CFTR, a hydrolytic event at the N-terminal
site opens the channel and another event at the C-terminal site closes
the channel. In ArsA, binding of ATP to the N-terminal site and its
hydrolysis occurs first and it is essential for participation of the
C-terminal NBD and for multisite catalysis.
 |
FOOTNOTES |
*
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.
To whom correspondence should be addressed. Tel.: 404-651-3864;
E-mail: pkaur@gsu.edu.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M211845200
 |
ABBREVIATIONS |
The abbreviations used are:
NBD, nucleotide
binding domain;
ATP
S, adenosine 5'-O-(thiotriphosphate);
FSBA, 5'-p-fluorosulfonylbenzoyladenosine;
MOPS, 4-morpholinepropanesulfonic acid.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.