Role of conserved aspartates in the ArsA ATPase.

Arsenic is widely distributed throughout the earth’s crust and is found in water that flowed through arsenic-rich rocks. Severe health effects have been observed in populations exposed to arsenic, which include cancer, cardiovascular disease, peripheral neuropathies and diabetes mellitus (1). Because of the prevalence of arsenic in the environment, nearly every organism, from Escherichia coli to humans, has genes for arsenic detoxification (2). The ArsAB pump in E. coli, encoded by the ars operon of plasmid R773, confers resistance to arsenicals and antimonials. ArsA1 is the catalytic subunit of the pump that hydrolyzes ATP in the presence of arsenite [As(III)] or antimonite [Sb(III)]. ATP hydrolysis is coupled to extrusion of As(III) or Sb(III) through ArsB, which serves both as a membrane anchor for ArsA and as the substrate-conducting pathway (3). Upon overexpression, ArsA exists primarily as a soluble protein in the cytosol. Purified ArsA is an As(III)/Sb(III)-stimulated ATPase (4). ArsA is composed of homologous N-terminal (A1) and C-terminal (A2) halves that are connected by a short linker (5). Each half contains a consensus sequence for a nucleotide-binding domain (NBD), and both NBDs are required for ATPase activity and metalloid transport (6, 7). The NBDs are formed by residues from both the A1 and A2 halves of the protein (8). A novel metalloid-binding domain (MBD), located at the A1–A2 interface diametrically opposite to the NBDs, allosterically modulates ATPase activity (9). The MBD contains one or more metalloid atoms that are coordinated to one residue from the A1 half and another from the A2 half of the protein (8). Thus metalloid serves as the “molecular glue” that holds the two halves of the protein together, forming the interface at the NBDs, activating catalysis. It has been proposed that conformational changes associated with nucleotide hydrolysis alter the affinities of the metalloid atoms bound at MBD, which are then injected into ArsB and extruded from the cell (8, 10). In the absence of metalloid, a condition that promotes basal hydrolysis, both NBD1 and NBD2 bind and hydrolyze ATP, albeit at a slow rate (11). In the presence of metalloid, ATP hydrolysis is stimulated in both NBD1 and NBD2, with hydrolysis at NBD1 stimulated to a greater degree than at NBD2 (11). These properties suggest that the two NBDs have different properties, and may therefore have different functions. The crystal structure of ArsA shows that Asp142 at NBD1 and Asp447 at NBD2 are in close proximity to the magnesium ions of the MgADP substrates (Figure 1). Asp142 is indirectly coordinated to the Mg2+ at NBD1 by its backbone carbonyl through a hydrogen bonded water molecule. Similarly, the Mg2+ at NBD2 is indirectly coordinated by water molecules that form hydrogen bonds with both the backbone and the carboxylate of Asp447 (8). In addition, each Mg2+ has a number of additional coordinations. The Mg2+ at NBD1 is coordinated to Thr22, Asp45, the β-phosphate of ADP, and indirectly to the α-phosphate of ADP through a water molecule. Asp45 has been previously identified as a Mg2+ ligand (12). The Mg2+ at NBD2 is coordinated to Thr341 and indirectly through water molecules to Ser363, Asp364, and the α- and β-phosphates of ADP. Structures of ArsA with ATP, the nonhydrolyzable ATP analogue, AMP-PNP, or the transition state analogue of ATPhydrolysis, ADP•AlF3, bound at NBD2 have also been determined. These structures indicate that binding of different nucleotides produces subtle distortions in the coordination geometry of Mg2+ at NBD2, which may be essential for ATP hydrolysis (10). Additionally, movement of amino acid side chains at NBD2 during ATP binding and hydrolysis produces large positional shifts at NBD1, indicating extensive crosstalk between NBD1 and NBD2 (10). Figure 1 Domains of the ArsA ATPase. The A1 and A2 STDs, two stretches of seven residues with the identical sequence D142/447TAPTGH148/453, connect the single MBD to the A1 and A2 MgADP-filled NBDs (8). The nucleotides bound in the two NBDs are shown as ball-and-stick ... Asp142 and Asp447 are located at the N-terminal end of an elongated stretch of residues, D142TAPTGH148 in A1 and D447TAPTGH453 in A2 forming the signal transduction domains (STDs) that physically connects the two NBDs to the single MBD and may be the primary effector of signal transduction between these centers (8). His148 and His453, located at the C-terminal end of the STDs, have been shown to be involved in transmission of information of metalloid occupancy in the single MBD to the two NBDs during catalysis (13), and in the crystal structure these two histidines can be seen to be ligands to two of the metalloids in the MBD (8), although the biochemical function of these two metalloids is not clear (9). The objective of this study was to determine what role the two aspartates (Asp142 and Asp447) play in Mg2+ binding, signal transduction, and catalysis. The results presented here suggest that the two aspartates have different functions.