From the Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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The ATPase activity of ArsA, the catalytic
subunit of the plasmid-encoded, ATP-dependent extrusion
pump for arsenicals and antimonials in Escherichia coli, is
allosterically activated by arsenite or antimonite. Magnesium is
essential for ATPase activity. To examine the role of
Asp45, mutants were constructed in which Asp45
was changed to Glu, Asn, or Ala. Cells expressing these mutated arsA genes lost arsenite resistance to varying degrees.
Purified D45A and D45N enzymes were inactive. The purified D45E enzyme exhibited approximately 5% of the wild type activity with about a
5-fold decrease in affinity for Mg2+. Intrinsic tryptophan
fluorescence was used to probe Mg2+ binding. ArsA
containing only Trp159 exhibited fluorescence enhancement
upon the addition of MgATP, which was absent in D45N and D45A. As
another measure of conformation, limited trypsin digestion was used to
estimate the surface accessibility of residues in ArsA. ATP and Sb(III)
synergistically protected wild type ArsA from trypsin digestion.
Subsequent addition of Mg2+ increased trypsin sensitivity.
D45N and D45A remained protected by ATP and Sb(III) but lost the
Mg2+ effect. D45E exhibited an intermediate
Mg2+ response. These results indicate that
Asp45 is a Mg2+-responsive residue, consistent
with its function as a Mg2+ ligand.
The ars operon of conjugative R-factor R773 encodes an
arsenite extrusion system that confers arsenite or antimonite
resistance to Escherichia coli by extruding arsenicals and
antimonials out of the cell, thus lowering their intracellular
concentration (1). This efflux pump is composed of two types of
subunits, ArsA and ArsB. The 63-kDa ArsA ATPase is the catalytic
subunit. The 45-kDa ArsB subunit is an integral membrane protein that
acts as the membrane anchor for ArsA and the oxyanion-translocating
sector of the pump (2). When overexpressed, ArsA is found in the
cytosol and purified as a soluble protein (3).
From its primary sequence, ArsA is composed of N-terminal (A1) and
C-terminal (A2) halves that are homologous to each other, most likely
the result of an ancestral gene duplication and fusion (4). Each half
contains a consensus sequence for the phosphate binding loop (P-loop)
of an ATP-binding site (5). Previous studies showed that both
ATP-binding sites are required for catalysis and resistance (6, 7). The
results of intergenic complementation and intragenic suppression
studies have suggested a model in which catalysis occurs at the
interface of the A1 and A2 ATP-binding sites (8). Recently, a highly
conserved DTAP consensus sequence has been identified in ArsA
homologues. These homologues are found in members of every kingdom from
bacteria to humans (9). From the results of intrinsic tryptophan
fluorescence, we have suggested this domain may act as a signal
transduction domain that relays the communication between ATP-binding
sites and the allosteric As(III)/Sb(III)-binding site.
From the sequence alignment of ArsA homologues with other enzymes such
as nitrogenase iron protein (NifH) (10), RecA (11), and GTP-binding
proteins, including Ras p21 (12), there is a highly conserved Asp-Pro
(Fig. 1), which, in G-proteins, forms part of an effector loop. From the crystal structure of Ras p21 with
GTP bound, the conserved Asp residue was shown to form a portion of the
Mg2+-binding site (12). In GTPases a magnesium ion brings
together diverse components of the GTP-binding core, facilitating the
information flow between domains. Elucidation of the Mg2+
coordination is therefore important for deciphering the mechanism of
nucleotide-binding proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Consensus sequences in ArsA homologues and
other nucleotide-binding proteins. The conserved P-loop and
Asp-Pro residues are shaded, and the position of
Asp45 is indicated with an asterisk. Shown are
the relevant sequences from the A1 and A2 halves of ArsA (J02591) and
from the ArsA homologues (Acidiphilium multivorum
(AB004659), Halobacterium sp. NRC1 plasmid NRC100
(AF016485), Methanococcus jannaschii (2128876),
Saccharomyces cerevisiae (S67642), Caenorhabditis
elegans (P30632), Chlorobium vibrioforme (U09867),
Aquifex aeolicus (AE000684), Homo sapiens
(U60276), Mus musculus (AF039405), and from other ATP- and
GTP-binding proteins NifH (U89346), Ras p21 (P01112), and RecA
(P03017)). Accession numbers are given in parentheses.
Sequence alignment of ArsA with GTPases and other ATPases suggests
that Asp45-Pro46 are the corresponding residues
in ArsA. In this study Asp45 was changed by mutagenesis to
several other residues. The properties of the resulting mutant strains
and the purified ArsAs support this postulate. First, an acidic residue
is required at position 45 for ArsA ATPase activity. The wild type
enzyme exhibited 20-fold more ATPase activity than the D45E derivative,
with 5-fold higher affinity for Mg2+. The D45A and D45N
derivatives were inactive. Second, using intrinsic tryptophan
fluorescence (13) and accessibility to trypsin to detect conformational
changes upon ligand binding (3), Asp45 was shown to be
required for Mg2+-dependent responses.
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MATERIALS AND METHODS |
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Medium and Growth Conditions-- E. coli strains and plasmids used in this study are described in Table I. Cells were grown in Luria-Bertani medium (14) at 37 °C. Ampicillin (125 µg/ml) and tetracycline (12.5 µg/ml) were added as required. Sodium arsenite was added at the indicated concentrations.
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DNA Manipulations-- The conditions for plasmid isolation, DNA restriction endonuclease analysis, ligation, and transformation have all been described (15). Restriction enzymes and nucleic acid-modifying enzymes were obtained from Life Technologies, Inc. The WizardTM plus minipreps DNA purification system and the WizardTM DNA clean-up system (Promega) were used to prepare plasmid DNA for restriction enzyme digestion and to recover DNA fragments from low melting agarose gels, respectively.
Oligonucleotide-directed Mutagenesis-- Mutations in the arsA gene were introduced by site-directed mutagenesis using the Altered SitesTM in vitro mutagenesis system (Promega) with plasmid pTZ3H6 containing the arsA and arsB genes (9) in E. coli ES1301 mutS. In this plasmid the arsA gene was previously mutated to contain only a single tryptophan codon (Trp159), and the sequence for six histidine codons was added at the 3' end. This plasmid was used as the template to produce arsA mutants with D45E, D45A, or D45N substitutions. The mutagenic oligonucleotides used and the respective changes (underlined) obtained were as follows: D45A, 5'-TGAGGCCGGAGCGGTACTGAC-3'; D45E, 5'-TTGAGGCCGGCTCGGTACTGA-3'; D45N, 5'-GAGGCCGGATTGGTACTGACC-3'.
All mutations were confirmed by sequencing the entire arsA gene using a Cy5-AUTOREAD sequencing kit with an ALFexpress system (Amersham Pharmacia Biotech). Plasmid DNA for sequencing was prepared with a Mini kit (QIAGEN).
Purification of ArsA--
Strain JM109 cells harboring the
indicated plasmids were grown at 37 °C in Luria-Bertani medium to
the mid-exponential phase of growth, at which point 0.1 mM
isopropyl -D-thiogalactopyranoside was added to induce
arsA expression. The cells were grown for another 2.5 h
before being harvested by centrifugation. The soluble ArsA proteins
were purified as described (9). ATPase activity was measured using an
NADH-coupled assay (16) with 5 mM ATP and 2.5 mM MgCl2 unless otherwise noted.
Fluorescence Measurements-- Fluorescence measurements were performed using an SLM-8000C spectrofluorometer with a built-in magnetic stirrer. The bandwidths for emission and excitation monochromators were 4 nm. Tryptophan fluorescence was monitored with an excitation wavelength of 295 nm and an emission wavelength of 337 nm. The fluorescence of the buffer (50 mM MOPS1-KOH, pH 7.5) alone was subtracted from protein spectra. ArsA (1.25 µM), ATP (5 mM), and MgCl2 (2.5 mM) were added as indicated.
Limited Trypsin Digestion of ArsA--
Limited trypsin digestion
was performed at room temperature and terminated at the indicated times
by the addition of a 3-fold excess of soybean trypsin inhibitor to the
reaction mixture as described previously (3). The reaction mixtures
were then analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (17).
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RESULTS |
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Effect of D45X Substitutions on Resistance to
Arsenite--
E. coli cells harboring plasmid pTZ3H6
exhibited resistance to 4 mM sodium arsenite (Fig.
2). The arsA gene in this
plasmid encodes a modified ArsA (W159H6) in which there is only a
single tryptophan residue at position 159 and six histidine residues fused to the C terminus. Resistance conferred by this plasmid was the
same as a plasmid bearing a wild type arsA gene (9), so this
modified arsA gene was used as the parent for all
D45X mutants. Mutational replacement of Asp45
with glutamic acid resulted in a reduction in arsenite resistance. Substitution of Asp45 with asparagine resulted in a further
decrease in resistance, and cells expressing a D45A substitution lost
arsenite resistance.
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ATPase Activity of D45X ArsAs-- The W159H6 ArsA and its D45X derivatives were purified by Ni2+ affinity chromatography to greater than 95% homogeneity, as judged by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue, and the ATPase activity of each protein was determined. In the presence of 5 mM ATP, 2.5 mM MgCl2, and 0.1 mM potassium antimonial tartrate, D45E ArsA exhibited approximately 5% of wild type ATPase activity (data not shown). In contrast, no activity was observed with either the D45A or D45N enzymes.
ATPase activity was measured at varying concentrations of
MgCl2. With the parental enzyme, the concentration of
MgCl2 required for half-maximal ATPase activity was 0.8 mM (Fig. 3). The activity of
the D45E enzyme was too low to obtain an accurate fit of the data, but
it required at least 5-fold more divalent cation for maximal activity
than the wild type. This suggests that the introduction of a glutamic
acid residue at position 45 lowered the affinity for Mg2+,
consistent with a role of Asp45 in coordination of the
divalent cation.
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Effect of Asp45 Substitutions on Intrinsic Fluorescence
of Trp159--
In the parental ArsA, Trp159
has a maximum emission at 337 nm in the native enzyme and at 353 nm in
the denatured protein when excited at 295 nm (9). Each of the
D45X proteins had the same emission maximum as the parental
(Fig. 4A), suggesting that the substitutions of Asp45 with Glu, Asn, or Ala per
se have little effect on the overall protein conformation.
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In the wild type ArsA the fluorescence of Trp159 has been shown to respond to MgATP binding (13). To produce an effect, both the divalent cation and ATP must be present (Fig. 4B). Compared with the parental enzyme, fluorescence of the D45E ArsA exhibited a smaller response to MgATP addition. With both parental and D45E enzymes the fluorescent enhancement produced by MgATP was reversed by the addition of disodium EDTA. In contrast, neither the D45A nor the D45N enzyme responded to the addition of Mg2+.
Effect of D45X Substitutions on Accessibility to
Trypsin--
Limited trypsin digestion has been used to study the
surface accessibility of arginine and lysine residues in the 63-kDa
ArsA (3). The initial cleavage by trypsin produces a fragment of approximately 50 kDa with subsequent cleavage to smaller polypeptides. The rate of cleavage was decreased synergistically by the binding of
both ATP and Sb(III), suggesting that the enzyme undergoes a
conformational change when both substrate and allosteric effector sites
are filled (13). However, those results were obtained in the absence of
Mg2+. The addition of Mg2+ proved to be
antagonistic to the protection conferred by ATP and Sb(III), increasing
the rate of trypsin cleavage (Fig. 5). This suggests that the binding of Mg2+ to the enzyme in
which the substrate and allosteric sites are already filled produces a
conformational change that makes a tryptic site more accessible.
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ATP and antimonite synergistically protected each D45X
protein to the same extent as the parental ArsA, indicating that
substitutions of Asp45 do not grossly affect the filling of
the allosteric and substrate sites (Fig. 5). However, Mg2+
caused no effect on the tryptic pattern in the D45A or D45N proteins and a partial response with the D45E ArsA. The antagonism by
Mg2+ on the parental and D45E ArsAs was reversed by EDTA,
indicating that the effect of Mg2+ requires an acidic
residue at position 45.
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DISCUSSION |
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Coordination of Mg2+ in ATP- and GTP-binding proteins frequently involves aspartate residues (18-21). ArsA, the catalytic subunit of the bacterial arsenite pump, hydrolyzes ATP to provide energy for arsenite efflux from cells (22). Mg2+ has been shown to be required for ArsA ATPase activity (3). The results described in this study indicate that the ArsA residue Asp45 is one ligand to the divalent cation.
First, sequence alignment of ArsA homologues demonstrates a conserved pair of residues that corresponds to Asp45-Pro46 in the R773 ArsA (Fig. 1). This DP sequence is conserved in other GTPases and ATPases, for example Asp33-Pro34 in Ras p21 (12), Asp100-Pro101 in RecA (11), and Asp39-Pro40 in the NifH iron protein subunit of nitrogenase (23) (Fig. 1). From the crystal structure of Ras p21 it was shown that in the GTP binding state Asp33 is a Mg2+ ligand. Similarly, from the crystal structure of NifH, Asp39 is in close proximity to the Mg2+ in the ATP-binding site. Based on the sequence comparisons, it is reasonable to hypothesize that Asp45 in ArsA may be involved in the coordination of the Mg2+ complexed with ATP.
Second, substitution of Asp45 in ArsA with Asn, Ala, or Glu resulted in reduced arsenite resistance for cells harboring the respective plasmid, indicating that an aspartate residue at position 45 promotes ArsA catalysis. Although an aspartate residue is not absolutely required, because the D45E and D45N mutants retained partial resistance, the wild type enzyme is approximately 20-fold more active than the purified D45E ArsA and has about a 5-fold higher affinity for Mg2+ (Fig. 3).
Third, Mg2+ produces conformational changes in ArsA that can be detected from the effect of the cation on the intrinsic fluorescence of Trp159 (Fig. 4) or on the surface accessibility of the protein to trypsin (Fig. 5). As shown previously, the fluorescence of Trp159 responds specifically to Mg2+ and ATP (13). With the D45E ArsA, the addition of Mg2+ and ATP induced an intermediate fluorescence enhancement that was reversed by EDTA. However, the fluorescence of the D45A or D45N proteins did not respond to MgATP. Moreover, the reversal of protection from trypsin digestion that Mg2+ afforded the wild type enzyme was absent in the D45N and D45A proteins and reduced in the D45E. These results strongly point to a role for Asp45 in binding Mg2+.
Several lines of evidence indicate that the substitutions did not
affect the overall conformation of the proteins or their ability to
bind the substrate ATP or the allosteric activator Sb(III). First, the
emission spectra of all of the proteins were superimposable, indicating
that the environment of Trp159 was the same in each (Fig.
4A). Second, the tryptic patterns of all the proteins were
similar in the absence of ligands (data not shown) or in the presence
of the nucleotide and the allosteric effector (Fig. 5), indicating that
the surface of the proteins had reasonably equal accessibility to large
molecules such as trypsin. The protection from trypsin afforded by the
combination of ATP and Sb(III) (but in the absence of Mg2+)
shows that the binding of a substrate or an allosteric effector was not
substantially altered by the replacement of Asp45 with
another acidic residue (glutamate), a neutral residue (asparagine), or
a smaller residue (alanine).
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant GM55425.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 reprint requests should be addressed: Dept. of
Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-1512; Fax: 313-577-2765; E-mail: brosen{at}med.wayne.edu.
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ABBREVIATIONS |
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The abbreviation used is: MOPS, 4-morpholinepropanesulfonic acid.
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