Nonequivalence of the Nucleotide Binding Domains of the ArsA ATPase

Abstract

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-[␣-32 P]-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 32 P at the ␥ 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.In Escherichia coli the ArsAB pump encoded by the ars operon of plasmid R773 confers resistance to arsenicals and antimonials (1). ArsA is the catalytic subunit of the pump that hydrolyzes ATP in the presence of the trivalent forms of the two metalloids. 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. Upon overexpression, ArsA exists primarily as a soluble protein in the cytosol. Soluble ArsA has been purified and characterized and has been shown to be an As(III)/Sb(III)-stimulated ATPase (2).ArsA is composed of homologous N-terminal (A1) and Cterminal (A2) halves that are connected by a short linker (3). Each contains a nucleotide binding domain (NBD) 1 (4) (Fig. 1), and both NBDs are required for ATPase activity and metalloid transport (5, 6). A novel metalloid binding domain located at the A1-A2 interface diametrically opposite to the NBDs modulates ATPase activity (4,7,8). Two stretches of residues, D 142 TAPTGH 148 in A1 and D 447 TAPTGH 453 in A2, physically connect the metalloid binding domain to the A1 and A2 NBDs, respectively. The metalloid binding domain is composed of three metalloid atoms, each of which is coordinated by two protein ligands, one of which is donated by A1 and the other by A2. Thus the metalloid atoms serve as the "molecular glue" that brings the two halves of the protein together, activating catalysis. The basal rate of catalysis has been thought to reflect the activity of onl...

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

“…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).…”

“…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.…”

“…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).…”

Role of Conserved Aspartates in the ArsA ATPase

“…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.…”

Mechanisms of Arsenic Detoxification and Resistance

“…These experiments suggested that the two homologous halves of ArsA are functionally nonequivalent (Jiang et al, 2005 (gray) are shown as space-filling models. A novel metalloid binding domain (MBD) is also located at the A1-A2 interface and is over 20 Å distant from the NBDs.…”