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J. Biol. Chem., Vol. 281, Issue 15, 9925-9934, April 14, 2006

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Cys-113 and Cys-422 Form a High Affinity Metalloid Binding Site in the ArsA ATPase^*

From the Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201

Received for publication, January 5, 2006 , and in revised form, February 1, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB extrusion pump for the trivalent metalloids As(III) and Sb(III). ArsA, the catalytic subunit has two homologous halves, A1 and A2. Each half has a consensus signal transduction domain that physically connects the nucleotide-binding domain to the metalloid-binding domain. The relation between metalloid binding by ArsA and transport through ArsB is unclear. In this study, direct metalloid binding to ArsA was examined. The results show that ArsA binds a single Sb(III) with high affinity only in the presence of Mg²⁺-nucleotide. Mutation of the codons for Cys-113 and Cys-422 eliminated Sb(III) binding to purified ArsA. C113A/C422A ArsA has basal ATPase activity similar to that of the wild type but lacks metalloid-stimulated activity. Accumulation of metalloid was assayed in intact cells, where reduced uptake results from active extrusion by the ArsAB pump. Cells expressing the arsA_C113A/C422AB genes had an intermediate level of metalloid resistance and accumulation between those expressing only arsB alone and those expressing wild type arsAB genes. The results indicate that, whereas metalloid stimulation of ArsA activity enhances the ability of the pump to reduce the intracellular concentration of metalloid, high affinity binding of metalloid by ArsA is not obligatory for transport or resistance. Yet, in mixed populations of cells bearing either arsAB or arsA_C113A/C422AB growing in subtoxic concentrations of arsenite, cells bearing wild type arsAB replaced cells with mutant arsA_C113A/C422AB in less than 1 week, showing that the metalloid binding site confers an evolutionary advantage.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The arsRDABC operon of the clinically isolated Escherichia coli plasmid R773 encodes the ArsAB ATPase, a metalloid pump that confers resistance by actively extruding As(III) or Sb(III) from cells (1). The 63-kDa ArsA is a metalloid-stimulated ATPase that comprises the catalytic subunit of the pump (2, 3). ArsA has two homologous halves, A1 and A2, connected by a short linker. Each half has a consensus nucleotide-binding domain (NBD).² In the presence or absence of the pump substrate, Sb(III) or As(III), both NBD1 and NBD2 hydrolyze ATP, with steady state hydrolysis dominated by the activity of NBD1 (4). The two NBDs are located at the interface between A1 and A2, in close proximity to each other (5). Over 20 Å distant from the NBDs is a metalloid-binding domain (MBD), where three Sb(III) or As(III) are bound at the A1-A2 interface (Fig. 1). One Sb(III) is connected to Cys-113 from A1 and Cys-422 from A2 (Site 1), a second to Cys-172 from A1 and His-453 from A2 (Site 2), and the third to His-148 from A1 and Ser-420 from A2 (Site 3). Thus, the three metalloid atoms act as molecular glue to bring the A1 and A2 halves of ArsA together, an event that is linked to activation of ATP hydrolysis. ArsA has two signature sequences that serve as signal transduction domains (STDs), D¹⁴²TAPTGH¹⁴⁸TIRLL in A1 (STD1) and D⁴⁴⁷TAPTGH⁴⁵³TLLLL in A2 (STD2), which corresponds to the Switch II region of many other nucleotide-binding proteins and have been proposed to be involved in transmission of the energy of ATP hydrolysis to metalloid transport (6). Asp-142 and Asp-447 are Mg²⁺ ligands in NBD1 and NBD2, respectively, and His-148 and His-453 are Sb(III) ligands in the MBD (5, 7).

The relation between binding of trivalent metalloid to ArsA and its transport through the ArsAB pump is not clear. Is the metalloid bound at the MBD a transport substrate or is it an allosteric activator of the pump? A C113A/C422A mutation introduced into ArsA was found to eliminate high affinity metalloid binding to ArsA. The C113A/C422A ArsA had basal ATPase activity similar to that of the wild type but lacked metalloid-stimulated activity. However, cells expressing the mutant ArsA_C113A/C422AB pump were able to extrude metalloid with higher efficiency than ArsB alone, exhibiting intermediate resistance between cells with wild type ArsAB and cells with only ArsB. These results indicate that the basal activity of the ArsAB pump is sufficient for ATP-driven efflux of metalloid and that the MBD is an allosteric site, with metalloid binding increasing pump activity. Finally, cells with wild type ArsAB were more successful than cells with the mutant ArsA_C113A/C422AB in growth in mixed cultures. We propose that in the low concentrations of metalloid ubiquitously present in the environment, the allosteric site evolved to increase the fitness of the organism for growth.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Media and Growth Conditions—The E. coli strains and plasmids used in this study are listed in Table 1. Cells were grown at 37 °C in Luria-Bertani (LB) medium (8) with either ampicillin (Ap) (125 µg/ml) or tetracycline (Tc) (12.5 µg/ml), as required. For arsenite resistance assays, overnight cultures were diluted 100-fold into fresh LB medium containing the indicated concentrations of sodium arsenite. After 6 h of growth at 37 °C, the absorbance at 600 nm was measured. Growth in mixed cultures was performed as follows. Cells of E. coli strain AW3110 bearing either pAlter1-ArsAB (Ap^r/Tc^s) or pAlter1-ArsA_C113A/C422AB (Ap^r/Tc^r) were grown overnight in LB medium at 37 °C. The cultures were mixed at a 1:1 ratio, diluted 1:1000 in fresh LB medium containing 125 µg/ml ampicillin and 4 µM sodium arsenite, and grown at 37 °C overnight. Each morning for 6 days the cultures were diluted 1:1000 into the same medium and grown overnight. Viable counts following serial dilutions were determined on LB plates containing 125 µg/ml ampicillin to obtain a total cell count or 12.5 µg/ml tetracycline to determine the fraction of cells bearing only pAlter1-ArsA_C113A/C422AB.

View larger version (44K): [in this window] [in a new window]   FIGURE 1. Domains of the ArsA ATPase. The A1 and A2 STDs (residues 142–148 and 447–453, respectively) connect the single MBD to the A1 and A2 MgADP-filled NBDs. Asp-142 and Asp-447 are coordinated to Mg2+ ions in NBD1 and NBD2, respectively. The MBD has three antimony atoms. The central metalloid atom is bound to the thiolates of Cys-113 and Cys-422, whereas the other two are liganded to harder atoms. The locations of tryptophan residues are indicated, both by the position in the ArsA sequence and as "molecular lanterns" that illuminate ArsA domains.

Purification of ArsA—Cells of strain JM109 bearing the indicated plasmids were grown at 37 °C in LB medium to mid-exponential phase, at which point 0.1 mM isopropyl -D-thiogalactopyranoside was added to induce arsA expression. The cells were grown for another 2.5 h, and then harvested by centrifugation. Soluble ArsA was purified as described (6). ATPase activity was measured using an NADH-coupled assay (10, 11) with 5 mM ATP and 2.5 mM MgCl₂, unless otherwise noted. The concentration of purified ArsA was determined using a Bio-Rad Protein Assay with a 340 PC microplate reader (Molecular Devices), using bovine serum albumin as a standard.

Measurement of Metalloid Binding—The buffer used for purification of ArsA was exchanged with a buffer containing 50 mM MOPS-KOH, pH 7.5 (Buffer A), using a Bio-Gel P-6 Micro Bio-Spin column (Bio-Rad). Purified protein was incubated at 4 °C with nucleotides, MgCl₂, and the indicated concentrations of potassium antimonyl tartrate. After 1 h, each sample was passed through a Bio-Gel P-6 column pre-exchanged with the Buffer A. Portions (25 µl) were diluted with 2% HNO₃, and the quantity of metalloid measured by inductively coupled mass spectrometry with a PerkinElmer ELAN 9000. Arsenic and antimony standard solutions in the range of 0.5–10 ppb in 2% HNO₃ were obtained from Ultra Scientific, Inc. (North Kingstown, RI).

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Stoichiometry of Sb(III) binding to ArsA. A, effect of nucleotides on the binding of Sb(III) to ArsA. 10 µM ArsA was incubated with 2.5 mM MgCl2, 2 m M nucleotide, and 100 µM antimonite. ATP, ATPS, ADP, no nucleotide, and 10 µM bovine serum albumin (BSA) incubated with 2.5 mM MgCl2 and 100 µM antimonite are indicated under the bars. The error bars represent the standard deviations (n = 4). B, antimonite binding to wild type (WT) and C113A/C422A ArsA. ArsA (10 µM) was incubated with 2.5 mM MgCl2, 2 mM ATPS, and increasing amounts of potassium antimonial tartrate. The lines represent best fit of the data using SigmaPlot 9.0 from five experiments for wild type ArsA and two experiments for the C113A/C422A ArsA. C, kinetic analysis of Sb(III) binding to either the wild type or C113A/C422A ArsA. Data were analyzed with the Scatchard equation: B = n-Kd·B/F, where B represents the concentration of Sb(III) bound to ArsA, and F represents the concentration of free Sb(III). Kd and n are the dissociation constant and the maximum number of Sb(III) bound per ArsA molecule, respectively.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. ATPase activity of ArsA. ATPase activity of either the wild type (WT) or C113A/C422A ArsA was determined in the presence of antimonite (A) or arsenite (B). The lines represent best fit of the data using SigmaPlot 9.0. The error bars represent the standard deviations (n = 3).

Transport Assays—Transport of arsenite by the ArsAB pump was assayed as decreased accumulation in cells (14, 15). Cells bearing plasmids were grown to A_{600 nm} of 1 at 37 °C with aeration in LB medium. The cells were harvested, washed, and suspended in a buffer consisting of 75 mM HEPES-KOH, pH 7.5, containing 0.15 M KCl and 1 mM MgCl₂ (Buffer B) and suspended at a density of 10¹⁰ cells/ml. To initiate the assay, 10 µM sodium arsenite, final concentration, was added in 1 ml of cells. Portions (0. 1 ml) were withdrawn at the indicated times, filtered through 0.45-µm pore diameter nitrocellulose filters (Whatman), and washed with 15 ml of the Buffer B, all at room temperature. The filters were dried and digested with 0.3 ml of 70% HNO₃ (EM Science) at 70 °C for 20 min, allowed to cool to room temperature, and diluted with 7 ml of high pressure liquid chromatography grade water (Sigma) to produce a final concentration of HNO₃ of 2.6%. Arsenic was quantified by inductively coupled mass spectrometry.

View larger version (69K): [in this window] [in a new window]   FIGURE 4. Limited trypsin digestion of ArsA. Purified wild type ArsA (panels A and B) or C113A/C422A ArsA (panels C and D) were treated with trypsin for various times without substrate or effector, with 0.5 mM potassium antimonyl tartrate, with 5 mM ATP, or with both ATP and potassium antimonyl tartrate, and analyzed by SDS-PAGE on 12% polyacrylamide gels stained with Coomassie Blue. Times of incubation are indicated on the top of each gel. The position of migration of standards is indicated.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

High affinity binding of Sb(III) to purified ArsA was measured by rapid gel filtration. Binding of As(III) could not be measured using this assay because of low affinity for this metalloid (cf. Fig. 3B). In the absence of nucleotide, only 0.1 mol of Sb(III) was bound per mol of ArsA (Fig. 2A). The same amount of Sb(III) binding was observed with bovine serum albumin, a protein of similar size to ArsA, indicating nonspecific interaction. Binding increased with addition of MgATP, MgADP, or MgATPS, with binding of 1 mol of Sb(III) per mol of ArsA with the nonhydrolyzable nucleotide (Fig. 2A). Reciprocally, binding of nucleotide is also enhanced by metalloid binding (4). However, it should be pointed out that the concentrations of ADP and ATP in the cytosol of E. coli are sufficient to saturate ArsA at all times, so in vivo binding of metalloid would be independent of nucleotide.

Binding to purified ArsA was measured as a function of Sb(III) concentration. In the absence of Mg²⁺-nucleotide, only background binding was observed. In the presence of saturating MgATPS, metalloid binding was saturable (Fig. 2B), with a stoichiometry of one Sb(III) per ArsA and an apparent K_d of 10^–5 M (Fig. 2C). In the presence of ATPS but in the absence of Mg²⁺, wild type ArsA still bound Sb(III) in a 1:1 ratio, but the apparent affinity was reduced by an order of magnitude (data not shown). In contrast, the C113A/C422A derivative bound only background levels of Sb(III) (Fig. 2, B and C), demonstrating that loss of Site 1 eliminates high affinity metalloid binding.

C113A/C422A ArsA Lacks Metalloid-stimulated ATPase Activity—The ability of Sb(III) to activate ArsA was examined in wild type and C113A/C422A ArsA (Fig. 3). The wild type enzyme exhibited a basal ATPase activity of 100 nmol mg^–1 min^–1. Both metalloids stimulated ATP hydrolysis but to different extents. Sb(III)-stimulated ATPase activity 8-fold (Fig. 3A), with half-maximal stimulation at 8 µM, a value similar to the K_d for binding of Sb(III) (Fig. 2C). As(III) stimulated activity 3-fold (Fig. 3B), with half-maximal stimulation at 340 µM. C113A/C422A ArsA had basal activity similar to that of the wild type but lacked metalloid-stimulated activity. These results show a clear relationship between binding of metalloid to the high affinity binding Site 1 and metalloid stimulation of catalytic activity.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Effect of ligands on the intrinsic fluorescence of W159 ArsA proteins. Fluorescence of W159 ArsA (A) and W159/C113A/C422A ArsA (B). At the arrows, 1 mM each, final concentration, of nucleotide, MgCl2 and potassium antimonyl tartrate were added sequentially. The excitation and emission wavelengths were 295 and 337 nm, respectively. WT, wild type.

Effect of Elimination of MBD Site 1 on NBD1—The fluorescence of several single tryptophan-containing ArsA derivatives has been shown to report conformational changes associated with ligand binding, with basal ATP hydrolysis, and with metalloid-stimulated ATPase activity (6, 9). Trp-159, which is located near the C-terminal end of STD1, reports slow conformational changes during basal hydrolysis and rapid conformational changes during metalloid-activated ATP hydrolysis (6). This fluorescence enhancement reports predominantly NBD1 activity in both the basal and activated states because NBD1 hydrolyzes ATP considerably faster than NBD2 under both sets of conditions (4). Moreover, the rate of fluorescence enhancement in the unactivated state is equivalent to the steady-state rate of basal hydrolysis (16). The effect of ligands on the intrinsic fluorescence of W159 ArsA was compared with a W159 derivative of C113A/C422A ArsA (Fig. 5). Both proteins exhibited slow enhancement of fluorescence in the presence of MgATP but not MgADP. Addition of Sb(III), which produces activated ATP hydrolysis, elicited a large and rapid quenching of fluorescence in the W159 enzyme (Fig. 5A) that was absent in the MBD Site 1 mutant (Fig. 5B). This is consistent with occupancy of MBD Site 1 by metalloid being required for activation of NBD1. When the rate of fluorescence enhancement was measured as a function of ATP concentration in W159 (Fig. 6A) or W159/C113A/C422A (Fig. 6B) ArsAs, no significant differences were observed. If Sb(III) is added to W159 ArsA prior to MgATP, no enhancement of intrinsic fluorescence is observed (17). In contrast, addition of Sb(III) to W159/C113A/C422A ArsA prior to MgATP did not prevent the nucleotide-dependent enhancement of fluorescence (Fig. 6C). The Site 1 mutant and its parent had similar affinity for ATP during basal hydrolysis, but Sb(III) did not increase the affinity of the mutant (Fig. 6D). Thus, MBD Site 1 appears to be required for activation of ATP hydrolysis but not for basal hydrolysis by NBD1.

Effect of MBD Site 1 on NBD2—Another single tryptophan derivative, M446W, has been useful for reporting events associated during nucleotide binding (9). The fluorescence of Trp-446, which is located between NBD2 and the first residue of the A2 STD (Fig. 1), has been shown to respond to binding of either MgATP or MgADP in the absence of metalloid with an enhancement of fluorescence. In the presence of metalloid there is rapid quenching of fluorescence with MgADP as the dinucleotide product fills NBD2. There is slower quenching with MgATP that reflects slow hydrolysis of the trinucleotide by NBD2, with concomitant filling of the binding site with the dinucleotide product. The rate of this quenching is much slower than the steady-state rate of ATP hydrolysis by ArsA, which is controlled by NBD1. Thus, the enhancement of Trp-446 fluorescence under unactivated conditions most likely reports nucleotide binding, and the slow rate of Trp-446 fluorescence quenching is constrained by the slow rate of hydrolysis in NBD2. The fluorescent properties of the M446W enzyme that were otherwise wild type and the C113A/C422A derivative were compared (Fig. 7). In the absence of metalloid, either MgATP or MgADP produced an enhancement of Trp-446 fluorescence with M446W ArsA (Fig. 7A). Subsequent addition of Sb(III) produced rapid quenching with MgADP and slow quenching with MgATP. The M446W/C113A/C422A ArsA exhibited a similar enhancement of fluorescence with either MgADP or MgATP (Fig. 7B), indicating that NBD2 binds nucleotide in the absence of MBD Site 1. In contrast, the mutant enzyme was unresponsive to the subsequent addition of Sb(III), consistent with a requirement for binding of Sb(III) to Site 1 to activate hydrolysis in NBD2.

Trp-461 Reports Binding of Metalloid to MBD Site 1—A new single tryptophan derivative, T461W, was constructed in an otherwise wild type ArsA background as a probe of metalloid binding to MBD Site 1. The intent was to alter Tyr-464, the A2 residue corresponding to Trp-159, to a tryptophan that could report similar conformational changes in A2. However, the fluorescence of a Y464W single tryptophan derivative was unresponsive to ligands (data not shown). Residues 462–479 are not visible in the crystal structure, indicating that they are in a mobile region of the protein. The closest residue to Tyr-464 visible in the structure is Thr-461. This residue occupies a position in A2 roughly corresponding to that of Trp-159 in A1, with both residues located on the outer surface of the A1-A2 interface near the MBD. T461W ArsA denatured with 6 M guanidine HCl (Fig. 8A, curve 5) had a fluorescence emission spectrum similar to an equimolar concentration of free tryptophan (curve 6), with an emission maximum at 353 nm. Native T461W exhibited a substantial blue shift and fluorescence enhancement, with an emission maximum at 334 nm (curve 1). Addition of saturating MgATP, MgADP, MgATPS(curve 3 shows only MgATP), or Sb(III) (data not shown) had little effect on fluorescence. Addition of saturating amounts of both Sb(III) and any of the three Mg²⁺-nucleotides (curve 4 shows only MgATP) produced substantial quenching of fluorescence. There was no shift in the emission maximum wavelength with the addition of these ligands, suggesting that the polarity of the environment of the indole side chain of Trp-461 was not greatly altered. The quenching of fluorescence of Trp-461 was assayed as a function of Sb(III) concentration in the presence of saturating MgATP, MgADP, MgATPS (only MgADP is shown in Fig. 8B), yielding a concentration of 10 µM for the half-maximal response for each of the three nucleotides (Fig. 8C). This value is comparable with the K_d value obtained by direct binding assays (Fig. 2C). The fluorescence titrations exhibited an apparent sigmoidicity that was absent in the direct metal binding titrations, but the significance of this response is not known. Perhaps the two weak binding sites contribute to the overall fluorescence response, a possibility that is being investigated. The fluorescence properties of a single T461W/C113A/C422A derivative were compared with T461W ArsA (Fig. 9). In the presence of either MgADP or MgATP, Sb(III) produced substantial fluorescence quenching in T461W ArsA (Fig. 9A). In contrast, there was little change in fluorescence with the T461W/C113A/C422A ArsA (Fig. 9B). Thus the synergistic response of Trp-461 to nucleotide and metalloid requires metalloid binding at Site 1 of the MBD.

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Effect of nucleotides on the intrinsic fluorescence of W159 ArsA proteins. A, fluorescence response of W159 ArsA. At the arrows, 1 m M MgCl2 and increasing amounts of ATP were added sequentially; B, fluorescence response of W159 ArsA with C113A/C422A mutation. At the arrows, 1 mM MgCl2 and increasing amounts of ATP were added sequentially; C, fluorescence response of W159 ArsA with the C113A/C422A mutation. At the arrows, 1 m M potassium antimonyl tartrate, 1 mM MgCl2, and increasing amounts of ATP were added sequentially; D, effect of ATP on the relative fluorescence changes of W159 ArsA with and without the C113A/C422A mutation. Curve 1, W159; curve 2, W159/C113A/C422A ArsA; curve 3, W159/C113A/C422A ArsA in the presence of antimonite. The data were plotted as the percentage of the maximum increases in the fluorescence of ArsA before addition of ATP as a function of the added ATP concentrations. The lines represent best fit of the data to the Hill equation using SigmaPlot 9.0. The excitation and emission wavelengths were 295 and 337 nm, respectively. WT, wild type.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. Effect of ligands on the intrinsic fluorescence of M446W ArsA proteins. Fluorescence of M446W ArsA (A) and M446W/C113A/C422A ArsA (B). At the arrows, 1 m M each, final concentration, of ATP, MgCl2, and potassium antimonyl tartrate were added sequentially. The excitation and emission wavelengths were 295 and 331 nm, respectively. WT, wild type.

This result suggests that the loss of Site 1 and the inability to activate ArsA reduces but does not eliminate the ability of the ArsAB pump to extrude metalloid. To examine that possibility, the steady state level of As(III) accumulation was measured in cells expressing wild type and mutant arsA (Fig. 10B). The steady state level of As(III) in cells of E. coli strain JM109 with vector was reduced in cells expressing arsB. The intracellular level of As(III) in cells expressing wild type arsAB was considerably less than arsB alone. Cells with the mutated arsA_C113A/C422AB genes had a steady state level of intracellular As(III) that was lower than cells with arsB but statistically higher than cells expressing the wild type pump. Thus, it appears that Site 1 is not obligatory for ArsAB function but increases the efficiency of the pump.

Binding of As(III) to ArsA Confers an Evolutionary Advantage—The increase in resistance conferred by wild type ArsA compared with the mutant lacking MBD Site 1 is rather modest, with the greatest differences observed at concentrations of arsenite exceeding those found in all but the most highly contaminated corners of the world. Is high affinity metalloid binding at the MBD sufficient to confer an evolutionary advantage on the host organism? To examine this question, cells of E. coli strain AW3110 bearing either wild type arsAB or arsA_C113A/C422AB were allowed to compete with each other in a mixed culture for growth in the presence of 4 µM sodium arsenite. Whereas this is a subtoxic concentration, it is equivalent to 300 ppb, which is about the midrange of arsenic found in water from highly contaminated wells in Bangladesh (18). Each day the culture was diluted 1000-fold, and the relative amounts of cells with the wild type and mutant pumps were determined (Fig. 11). By 4 days of growth, cells with arsAB had largely replaced those with only arsA_C113A/C422AB. We propose that the MBD provides a competitive advantage to cells expressing the wild type ArsAB As(III)/Sb(III)-translocating ATPase for growth under environmental conditions where arsenic is commonly found.

View larger version (22K): [in this window] [in a new window]   FIGURE 8. Intrinsic fluorescence of T461W ArsA proteins. A, emission spectra of T461W ArsA. Curve 1, T461W; curve 2, T461W plus 1 mM ATP; curve 3, T461W plus 1 mM ATP and1mM MgCl2; curve 4, T461W plus 1 mM MgATP and 1 mM antimonite; curve 5, T461W plus 6 M guanidine HCl; curve 6, 1.25 µM tryptophan. B, fluorescence response of T461W ArsA. At the arrows, 1 m M ADP, 1 mM MgCl2, and increasing amounts of potassium antimonyl tartrate were added sequentially. The excitation and emission wavelengths were 295 and 334 nm, respectively. C, effect of antimonite on the relative fluorescence changes of T461W ArsA in the presence of different Mg-nucleotides. The data were plotted as the percentages of the quench in the fluorescence of ArsA before addition of antimonite as a function of the added concentrations of potassium antimonyl tartrate. The lines represent best fit of the data to the Hill equation using SigmaPlot 9.0. The error bars represent the standard deviations (n = 3). WT, wild type.

View larger version (19K): [in this window] [in a new window]   FIGURE 9. Effect of ligands on the intrinsic fluorescence of T461W ArsA proteins. Fluorescence of T461W ArsA (A) and mutant T461W/C113A/C422A ArsA (B). At the arrows, 1 m M each, final concentration, of ATP, MgCl2, and potassium antimonyl tartrate were added sequentially. The excitation and emission wavelengths were 295 and 334 nm, respectively. WT, wild type.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (23K): [in this window] [in a new window]   FIGURE 10. Resistance to arsenite in cells expressing wild type and mutant arsA genes. A, overnight cultures of E. coli strain JM109 bearing wild type and mutant ars plasmids were diluted 100-fold into fresh LB medium containing various concentrations of sodium arsenite and the cell density was measured after 6 h of growth at 37 °C. B, arsenite uptake into cells of E. coli strain JM109. Transport of arsenite was assayed as described under "Experimental Procedures." Cells had the following plasmids: pArsAB •; pArsAC113A/C422AB ; pArsB, ; and vector plasmid, pAlter-1, . The error bars represent the standard deviations (n = 3).

View larger version (14K): [in this window] [in a new window]   FIGURE 11. The high affinity metalloid binding site of ArsA increases the ability of cells to compete in an arsenic-containing environment. Competition assays between cells with arsAB and arsAC113A/C422AB were performed as described under "Experimental Procedures." Mixed cultures of cells of E. coli strain AW3110 bearing either pArsAB or pArsAC113A/C422AB were allowed to grow with daily dilutions into fresh medium. By 4 to 6 days, cells with pArsAC113A/C422AB were lost from the population, as determined from the fraction of Tcr colonies. The data are the average of two separate assays.

A second single tryptophan derivative containing only Trp-446, which is located adjacent to NBD2, reports binding of MgADP and MgATP in the absence of metalloid with a slight enhancement of fluorescence (6). This nucleotide-dependent enhancement of Trp-446 fluorescence was unaffected by mutagenesis of Site 1 (Fig. 7B). When Sb(III) was added to ArsA containing bound MgADP, there was a rapid quenching of fluorescence (Fig. 7A) that corresponds to an increase in affinity for nucleotide (4). In contrast, when Sb(III) was added to the Trp-446 ArsA with bound MgATP, there was a slow quenching that corresponds to the rate of formation of ADP by NBD2. This reflects the fact that the two NBDs are not equivalent, and NBD2 is always slower than NBD1, even in the activated state (4). Thus, loss of Site 1 does not affect binding of nucleotide or basal catalysis in NBD2 but does prevent activation of catalysis in NBD2. Overall, the data indicate that Site 1 is required for activation of catalysis in both NBD1 and NBD2 by metalloid.

A fundamental issue is the role of the MBD in metalloid transport and resistance. One possibility is that the MBD is a transport site in which the bound metalloid atom is inserted into the translocation channel of ArsB as a result of ATP hydrolysis. The results of mutagenesis argue against obligatory binding at Site 1 for physiological function. Cells expressing the arsA_C113A/C422AB genes had reduced transport and resistance compared with cells expressing wild type arsAB, even though purified C113A/C422A ArsA lost high affinity metalloid binding. We cannot rule out the possibility that the metalloid bound at Site 1 is a transport substrate but that alternate pathways, perhaps through Sites 2 or 3, assume that role in the mutant. A more sensible inference is that Site 1 is an allosteric site that enhances the activity of the pump without contributing its bound metalloid to ArsB. Finally, why did the MBD evolve? Clearly cells with the plasmid-encoded ArsAB pump are considerably more resistant to arsenite than cells that express only chromosomal ArsB (15, 21). But is the presence of Site 1, which confers only a small growth advantage, sufficient to confer a competitive advantage over cells expressing an ArsA lacking the Cys-113 and Cys-422? When strains with wild type and mutant pumps were allowed to compete with each other in media containing a subtoxic concentration of arsenite, those with the wild type ArsA easily outgrew the mutants. Thus, the ability to activate ArsA by binding of metalloid to the MBD gives increased fitness for growth in containing concentrations of arsenic found in drinking water in many parts of the world (18).

	FOOTNOTES

 
^* 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 313-577-1512; Fax: 313-577-2765; E-mail: brosen{at}med.wayne.edu.

² The abbreviations used are: NBD, nucleotide-binding domain; MBD, metalloid-binding domain; STD, signal transduction domain; Ap, ampicillin; Tc, tetracycline; ATPS, adenosine 5'-O-(3-thiotriphosphate); MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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