Tongqing Zhou

Structure of the ArsA ATPase: the catalytic subunit of a heavy metal resistance pump

Active extrusion is a common mechanism underlying detoxification of heavy metals, drugs and antibiotics in bacteria, protozoa and mammals. In Escherichia coli, the ArsAB pump provides resistance to arsenite and antimonite. This pump consists of a soluble ATPase (ArsA) and a membrane channel (ArsB). ArsA contains two nucleotide‐binding sites (NBSs) and a binding site for arsenic or antimony. Binding of metalloids stimulates ATPase activity. The crystal structure of ArsA reveals that both NBSs and the metal‐binding site are located at the interface between two homologous domains. A short stretch of residues connecting the metal‐binding site to the NBSs provides a signal transduction pathway that conveys information on metal occupancy to the ATP hydrolysis sites. Based on these structural features, we propose that the metal‐binding site is involved directly in the process of vectorial translocation of arsenite or antimonite across the membrane. The relative positions of the NBS and the inferred mechanism of allosteric activation of ArsA provide a useful model for the interaction of the catalytic domains in other transport ATPases.

Tryptophan Fluorescence Reports Nucleotide-induced Conformational Changes in a Domain of the ArsA ATPase

The ars operon of plasmid R773 encodes an ATP-dependent extrusion pump for arsenite and antimonite in Escherichia coli. The ArsA ATPase is the catalytic subunit of the pump protein, with two nucleotide binding consensus sequences, one in the NH2-terminal half and one in the COOH-terminal half of the protein. A 12-residue consensus sequence (DTAPTGHTIRLL) has been identified in ArsA homologs from eubacteria, archebacteria, fungi, plants, and animals. ArsA enzymes were constructed containing single tryptophan residues at either end of this conserved sequence. The emission spectrum of the fluorescence of the tryptophan on the COOH-terminal end (Trp-159) indicated a relatively hydrophilic environment for this residue. An increase in intrinsic tryptophan fluorescence and a blue shift of the maximum emission wavelength were observed upon addition of MgATP, indicating movement of Trp-159 into a relatively less polar environment. No fluorescence response was observed with MgADP, with nonhydrolyzable ATP analogs, or with MgATP by catalytically inactive enyzmes. This suggests that the location Trp-159 is shifted only during hydrolysis of ATP. In contrast, the emission spectrum of Trp-141, located on the NH2-terminal side of the consensus sequence, indicated a relatively nonpolar environment. The maximum emission wavelength red shifted upon addition of MgADP. MgATP slowly produced a response that correlated with product formation, suggesting that the environment of Trp-141 is sensitive only to MgADP binding. Thus, during ATP hydrolysis the COOH-terminal end of the conserved domain moves into a less polar environment, whereas the NH2-terminal end moves into a more hydrophilic environment as product is formed. A hypothesis is presented in which the conserved domain of ArsA and homologs is an energy transduction domain involved in transmission of the energy of ATP hydrolysis to biological functions such as transport.

Mechanism of the ArsA ATPase.

The ArsAB ATPase confers metalloid resistance in Escherichia coli by pumping toxic anions out of the cells. This transport ATPase shares structural and perhaps mechanism features with ABC transporters. The ArsAB pump is composed of a membrane subunit that has two groups of six transmembrane segments, and the catalytic subunit, the ArsA ATPase. As is the case with many ABC transporters, ArsA has an internal repeat, each with an ATP binding domain, and is allosterically activated by substrates of the pump. The mechanism of allosteric activation of the ArsA ATPase has been elucidated at the molecular level. Binding of the activator produces a conformational change that forms a tight interface of the nucleotide binding domains. In the rate-limiting step in the overall reaction, the enzyme undergoes a slow conformational change. The allosteric activator accelerates catalysis by increasing the velocity of this rate-limiting step. We postulate that similar conformational changes may be rate-limiting in the mechanism of ABC transporters.

The ATPase Mechanism of ArsA, the Catalytic Subunit of the Arsenite Pump

The ArsA ATPase is the catalytic subunit of a novel arsenite pump, with two nucleotide-binding consensus sequences in the N- and C-terminal halves of the protein. The single tryptophan-containing Trp159 ArsA was used to elucidate the elementary steps of the ATPase mechanism by fluorescence stopped-flow experiments. The binding and hydrolysis of MgATP is a multistep process with a minimal kinetic mechanism (Mechanism 1). Mechanism 1 A notable feature of the reaction is that MgATP binding induces a slow transient increase in fluorescence of ArsA, which is independent of the ATP concentration, indicative of the build-up of a pre-steady state intermediate. This finding, coupled with a phosphate burst, implies that the steady-state intermediate builds up subsequent to product release. We propose that the rate-limiting step is an isomerization between different conformational forms of ArsA.k cat is faster than the phosphate burst, indicating that both nucleotide binding sites of ArsA are catalytic. Consistent with this interpretation, approximately 2 mol of phosphate are released per mole of ArsA during the phosphate burst.

Asp45 is a Mg2+ ligand in the ArsA ATPase.

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 Asp45was changed to Glu, Asn, or Ala. Cells expressing these mutatedarsA 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.

Unisite and multisite catalysis in the ArsA ATPase.

The ars operon of plasmid R773 encodes an As(III)/Sb(III) extrusion pump. The catalytic subunit, the ArsA ATPase, has two homologous halves, A1 and A2, each with a consensus nucleotide-binding sequence. ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. ArsA M446W has a single tryptophan adjacent to the A2 nucleotide-binding site. Tryptophan fluorescence increased upon addition of ATP, ADP, or a nonhydrolyzable ATP analogue. Mg2+ and Sb(III) produced rapid quenching of fluorescence with ADP, no quenching with a nonhydrolyzable analogue, and slow quenching with ATP. The results suggest that slow quenching with ATP reflects hydrolysis of ATP to ADP in the A2 nucleotide-binding site. In an A2 nucleotide-binding site mutant, nucleotides had no effect. In contrast, in an A1 nucleotide-binding mutant, nucleotides still increased fluorescence, but there was no quenching with Mg2+ and Sb(III). This suggests that the A2 site hydrolyzes ATP only when Sb(III) or As(III) is present and when the A1 nucleotide-binding domain is functional. These results support previous hypotheses in which only the A1 nucleotide-binding domain hydrolyzes ATP in the absence of activator (unisite catalysis), and both the A1 and A2 sites hydrolyze ATP when activated (multisite catalysis).

Nonequivalence of the nucleotide binding domains of the ArsA ATPase

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB pump that catalyzes extrusion of the metalloids As(III) and Sb(III), conferring metalloid resistance. The catalytic subunit, ArsA, is an ATPase with two homologous halves, A1 and A2, connected by a short linker. Each half contains a nucleotide binding domain. The overall rate of ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. The results of photolabeling of ArsA with the ATP analogue 8-azidoadenosine 5′-[α-32P]-triphosphate at 4 °C indicate that metalloid stimulation correlates with a >10-fold increase in affinity for nucleotide. To investigate the relative contributions of the two nucleotide binding domains to catalysis, a thrombin site was introduced in the linker. This allowed discrimination between incorporation of labeled nucleotides into the two halves of ArsA. The results indicate that both the A1 and A2 nucleotide binding domains bind and hydrolyze trinucleotide, even in the absence of metalloid. Sb(III) increases the affinity of the A1 nucleotide binding domain to a greater extent than the A2 nucleotide binding domain. The ATP analogue labeled with 32P at the γ position was used to measure hydrolysis of trinucleotide at 37 °C. Under these catalytic conditions, both nucleotide binding domains hydrolyze ATP, but hydrolysis in A1 is stimulated to a greater degree by Sb(III) than A2. These results suggest that the two homologous halves of the ArsA may be functionally nonequivalent.

Antimonite regulation of the ATPase activity of ArsA, the catalytic subunit of the arsenical pump.

The ArsA ATPase is the catalytic subunit of the pump protein, coupling the hydrolysis of ATP to the movement of arsenicals and antimonials through the membrane-spanning ArsB protein. Previously, we have shown the binding and hydrolysis of MgATP to ArsA to be a multi-step process in which the rate-limiting step is an isomerization between different conformational forms of ArsA. This isomerization occurs after product release, at the end of the ATPase reaction, and involves the return of the ArsA to its original conformation, which can then bind MgATP. ArsA possesses an allosteric site for antimonite [Sb(III)], the binding of which elevates the steady-state ATPase activity. We have used a transient kinetics approach to investigate the kinetics of ternary complex formation that lead to an enhancement in the ATPase activity. These studies revealed that ArsA exists in at least two conformational forms that differ in their ligand binding affinities, and that ATP favours one form and Sb(III) the other. Ternary complex formation is rate-limited by a slow transition between these conformational forms, leading to a lag in attaining maximal steady-state activity. Sb(III) enhances the steady-state ATPase activity by inducing rapid product release, allowing ArsA to adopt a conformation that can bind MgATP for the next catalytic cycle. In the presence of Sb(III), ArsA avoids the rate-limiting isomerization at the end of the ATPase reaction and ATP hydrolysis becomes rate-limiting for the reaction. The binding of Sb(III) probably results in more effective pumping of the substrates from the cell by enhancing the rate of efflux.