Steric limitations in the interaction of the ATP binding domains of the ArsA ATPase.

ArsA, the catalytic subunit of an anion-translocating ATPase, has two consensus nucleotide binding sites, one N-terminal and one C-terminal. A mutation producing a G15C substitution in the N-terminal domain resulted in substantial reductions in arsenite resistance, transport, and ATPase activity. A second site revertant (A344V) adjacent to the C-terminal nucleotide binding site was previously shown to restore arsenite resistance, suggesting the interaction of the nucleotide binding sites in ArsA (Li, J., Liu, S., and Rosen, B. P. (1996) J. Biol. Chem. 271, 25247-25252). In this study, it is shown that alteration of Ala-344 to bulkier residues, including Cys, Thr, Pro, Asp, Leu, Phe, Tyr, or Arg, also suppressed the G15C substitution. However, A344G or A344S substitutions only marginally suppressed the primary mutation. Alteration of Gly-15 to Ala, Cys, Asp, Tyr, or Arg each resulted in decreased arsenite resistance. The larger the residue volume of the substitution, the lower the resistance, with a G15R substitution producing the least resistance. Resistance in a strain expressing an arsA gene encoding the G15R substitution could be rescued by A344S, A344T, A344D, A344R, or A344V second site suppressors. The larger the residue is then the greater the suppression is. The in vitro ArsA ATPase activities from purified wild type, G15A, G15C, and G15R exhibits an inverse relationship between activity and residue volume. Purified G15A and G15C exhibited both an increase in the Km for ATP and a decrease in Vmax. The results are consistent with a physical interaction of the two nucleotide binding domains and indicate that the geometry at the interface between the N- and C-terminal nucleotide binding sites places spatial constraints on allowable residues in that interface.