An arsenic metallochaperone for an arsenic detoxification pump

Abstract

Environmental arsenic is a world-wide health issue, making it imperative for us to understand mechanisms of metalloid uptake and detoxification. The predominant intracellular form is the highly mephitic arsenite, which is detoxified by removal from cytosol. What prevents arsenite toxicity as it diffuses through cytosol to efflux systems? Although intracellular copper is regulated by metallochaperones, no chaperones involved in conferring resistance to other metals have been identified. In this article, we report identification of an arsenic chaperone, ArsD, encoded by the arsRDABC operon of Escherichia coli. ArsD transfers trivalent metalloids to ArsA, the catalytic subunit of an As(III)/Sb(III) efflux pump. Interaction with ArsD increases the affinity of ArsA for arsenite, thus increasing its ATPase activity at lower concentrations of arsenite and enhancing the rate of arsenite extrusion. Cells are consequently resistant to environmental concentrations of arsenic. This report of an arsenic chaperone suggests that cells regulate the intracellular concentration of arsenite to prevent toxicity.

Fig. 1.

ArsD enhances the activity of the ArsAB pump in vivo. (A) Molecular competition between cells with arsDAB and arsAB. Cells of E. coli strain AW3110 bearing either pSE-AB or pSE-DAB were grown separately or in mixed culture of each for 9 days with daily dilutions into fresh LB medium containing 10 μM sodium arsenite, as described in Materials and Methods. The plasmids from equal amounts of cells were extracted and digested with restriction enzymes XbaI and BamHI, and equal amounts of DNA containing the restriction fragments were separated by electrophoresis on a 1% agarose gel. Both plasmids produced a large fragment (data not shown) of vector DNA, and both produced a small arsB-containing BamHI fragment of 1.1 kb (fragment Z). Digestion of pSE-AB also produced a 1.6-kb fragment containing most of arsA (fragment Y), and digestion of pSE-DAB yielded the 1.9-kb fragment X containing arsD and most of arsA. The restriction fragments were from cells with only pSE-AB before growth (lane 1) or after 9 days (lane 2), only pSE-DAB before growth (lane 3) or after 9 days (lane 4), or from a mixed culture of cells with either pSE-AB or pSE-DAB before growth (lane 6) or after 3 days (lane 7), 6 days (lane 8), or 9 days (lane 9). Lane 5 contains a standard of λ HindIII-digested DNA. (B) Cells with only arsAB are lost from the population. The fraction of each plasmid was calculated by quantifying bands X, Y, and Z by densitometry. The percentage of the cells with each plasmid was calculated as follows. arsDAB: X/((vector + Z)/2); arsAB: Y/((vector + Z)/2). The data are the mean of values from five separate gels representing three independent experiments. The error bars represent the standard deviation of the mean calculated by using SigmaPlot 9.0. (C) Cells expressing arsDAB maintain lower intracellular arsenite than do cells expressing arsAB. Transport of As(III) was assayed with AW3110 bearing vector plasmids pSE380 and pACBAD (▵) or plasmids with arsB (○), arsAB (▿), arsD and arsAB (□), or arsD and arsB (◇).

Fig. 2.

ArsD and ArsA interact in vivo and in vitro. (A) Yeast two-hybrid assays with wild-type ars genes. Yeast strain AH109 bearing both GAL4 AD and BD fusion plasmids was grown in SD medium overnight and inoculated on agar plates with SD lacking histidine with 10-fold serial dilutions. The plates were incubated at 30°C for 2–3 days. As a positive control, pVA3 (BD-p53) was expressed with pTD1 (AD-T antigen); as a negative control, vector plasmid pGBT9 was expressed with pACT2 (alternating in the top row). (B) bBBr cross-linking. The indicated proteins (16 μM concentrations each) were incubated with 0.5 mM bBBr and/or 1 mM concentrations each of potassium antimonyl tartrate, MgCl₂, and ATP. Samples were analyzed by SDS/PAGE and immunoblotting with anti-ArsA (Top), anti-ArsD (Middle), or anti-CadC (Bottom). Lane 1, ArsA + ArsD; lane 2, ArsA + ArsD + bBBr; lane 3, ArsA + ArsD + Sb(III) + bBBr; lane 4, ArsA + ArsD + MgATP + bBBr; lane 5, ArsA + ArsD + Sb(III) + MgATP + bBBr; lane 6, ArsA + CadC + bBBr; lane 7, ArsA + CadC + MgATP + bBBr; lane 8, ArsA + CadC + Sb(III) + MgATP + bBBr. The position of individual proteins is indicated by arrows. The location of the band that reacts with both antibodies (the putative ArsD–ArsA adduct) is indicated with an asterisk.

Fig. 3.

ArsD transfers metalloid to and activates the ArsA ATPase. (A) ArsA releases Sb(III) from ArsD. Sb(III)–MBP–ArsD was bound to a 2-ml amylose column. One milliliter of either 20 μM ArsA or BSA preincubated with 1 mM MgCl₂ or MgATP was applied to the column. The column was washed with 8 ml of column buffer, and MBP–ArsD was eluted with 4 ml of 10 mM maltose. The protein in each fraction was identified by SDS/PAGE. BSA or ArsA eluted primarily in fraction 2, and most of the MBP–ArsD eluted in fraction 11. The molar concentration of each protein in the fractions was estimated from the absorption at 280 nm (white bars), and amount of Sb(III) was determined by inductively coupled plasma mass spectroscopy (black bars). (B) A nucleotide enhances the ArsA-induced release of Sb(III) from ArsD. Sb(III) release from ArsD was assayed in the presence of the indicated nucleotides and is expressed relative to the values with BSA. The values are the average of two independent assays. (C) ArsD transfers As(III) to ArsA. The molar ratio of As(III) to either ArsA or ArsD monomer was measured with protein either alone (black bars) or in the presence of the partner protein (white bars). The values are the mean of three independent assays. (D) The transfer of Sb(III) from ArsD to ArsA time-resolved by stopped-flow fluorescence spectroscopy. Equal volumes of the following reagents were mixed in a stopped-flow device, and the changes in protein fluorescence (excitation = 285 nm; emission >340 nm) monitored. Curve A, 2 μM ArsD + 10 μM ArsA; curve B, 2 μM ArsD/10 μM Sb(III) + 10 μM ArsA; curve C, 10 μM ArsA + buffer; curve D, 10 μM ArsA + 10 μM Sb(III); curve E, 2 μM ArsD + 10 μM Sb(III); curve F, 2 μM ArsD/10 μM Sb(III) + buffer. Each division on the y axis represents a fluorescence change of 5% relative to that of 2 μM ArsD/10 μM ArsA.

Fig. 4.

ArsD increases the affinity of the ArsA ATPase for As(III). (A) ArsD lowers the K_1/2 of ArsA for As(III). ATPase activities were measured in the absence and presence of MBP–ArsD at varying concentrations of sodium arsenite. (B) ArsD does not affect the affinity of ArsA for ATP. ArsA ATPase activities were measured at varying concentrations of ATP in the presence of 0.5 mM sodium arsenite and the presence or absence of ArsD. The values in each plot are the mean of three independent assays. The error bars represent the standard deviation of the mean, which were calculated by using SigmaPlot 9.0, as were the V_max, K_1/2, and K_m values.