Ligand interactions in the ArsA protein, the catalytic component of an anion-translocating adenosinetriphosphatase

Abstract

The ars operon of the conjugative R-factor R773 produces resistance to arsenicals in cells of Escherichia coli. The operon encodes an oxyanion pump which is composed of a membrane subunit, the 45.5-kDa ArsB protein, and a catalytic subunit, the 63-kDa ArsA protein. Purified ArsA protein is an arsenite(antimonite)-stimulated ATPase. From its amino acid sequence, as deduced from the nucleotide sequence, the ArsA protein has four tryptophanyl residues which could serve as intrinsic fluorescent probes for the study of substrate-induced conformational changes. Both static and dynamic measurements of tryptophan fluorescence were performed with the ArsA protein. Results from static anisotropy measurements indicated differences in molecular motion with addition of ATP, SbO2-, or Mg2+. These results were supported by time decay measurements of fluorescence anisotropy. The results of time decay measurements indicated a shorter correlation time, reflecting localized motion in the vicinity of the probe, and a longer correlation time, which could have arisen from rotation of the major portion of the molecule. The longer correlation time changed with addition of the various effectors, especially MgCl2, suggesting that binding of Mg2+ decreases probe mobility.

Substrate-Dependent Conformational Dynamics of the Escherichia coli Membrane Insertase YidC

“…In particular, for the residues W332/W334 (2W) and W508 these changes in mobility are concomitant with an explicit increase of the corresponding correlation time. Similar behavior upon ligand binding, i.e., an increase of segmental mobilty, was reported in other protein−ligand interaction studies using time-resolved Trp fluorescence anisotropy. , …”

Gene regulation of plasmid- and chromosome-determined inorganic ion transport in bacteria.

“…The ArsA ATPase subunit has been purified and characterized (84,(95)(96)(97). It is an arsenite-(and antimonate-) stimulated ATPase (182).…”

“…One region may be enzymatic and the other regulatory. An interaction between the two subunits of an ArsA dimer and the ATP-binding sites is indicated by the finding that the binding of only one fluorosulfonylbenzoyladenoside molecule per dimer was sufficient to inactivate ATPase activity in the presence of antimonite (83,97).…”

An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in gram-negative bacteria

“…In the wellstudied ars-containing plasmid R773, isolated from Escherichia coli (7,34), the operon consists of five genes that are controlled from a single promoter located upstream of the first cistron (arsR). These cistrons, arsRDABC (in that order), encode an arsenic-inducible repressor (arsR) (46), a negative regulatory protein that controls the upper level of transcription (arsD) (48), an ATPase plus membrane-located arsenite efflux pump (arsA and arsB, respectively) (20,29,35,42), and an arsenate reductase (arsC) (18). In the well-studied ars-containing plasmids isolated from Staphylococcus species (plasmids pI258 and pSX267), the arsR, arsB, and arsC cistrons are conserved, while the arsD and arsA cistrons are absent (17,30).…”