Mutations in the ArsA ATPase that restore interaction with the ArsD metallochaperone
The ArsA ATPase is the catalytic subunit of the ArsAB As(III) efflux pump. It receives trivalent As(III) from the intracellular metallochaperone ArsD. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. A number of arsA genes with multiple mutations were isolated. These were analyzed in more detail by separation into single arsA mutants. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant in yeast two-hybrid assays. Each of the three single ArsA mutants also interacted with wild type ArsD. Only the Q56R ArsA derivative exhibited significant metalloid-stimulated ATPase activity in vitro. Purified Q56R ArsA was stimulated by wild type ArsD and to a lesser degree by the quadruple ArsD derivative. The F120I and D137V ArsAs did not show metalloid-stimulated ATPase activity. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. We predict that mutations in ArsA propagate changes in hydrogen bonding and salt bridges to the ArsA–ArsD interface that affect their interactions.
KeywordsArsenite ArsA ArsD Metallochaperone Resistance Transport ATPase
This study was supported by National Institutes of Health Grant GM55425.
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