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 res ulted in substantial reductions in arsenite resistance, transport, and ATPase activity. A second site revertant (A344V) adjacent to the C-te rminal nucleotide binding site was previously shown to restore arsenit e resistance, suggesting the interaction of the nucleotide binding sit es in ArsA (Li, J., Liu, S., and Rosen, B. P. (1996) J. Biol. Chem. 27 1, 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 su bstitutions only marginally suppressed the primary mutation. Alteratio n of Gly-15 to Ala, Cys, Asp, Tyr, or Arg each resulted in decreased a rsenite resistance. The larger the residue volume of the substitution, the lower the resistance, with a G15R substitution producing the leas t 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 g reater the suppression is. The in vitro ArsA ATPase activities from pu rified wild type, G15A, G15C, and G15R exhibits an inverse relationshi p between activity and residue volume. Purified G15A and G15C exhibite d both an increase in the K-m for ATP and a decrease in V-max. The res ults are consistent with a physical interaction of the two nucleotide binding domains and indicate that the geometry at the interface betwee n the N- and C-terminal nucleotide binding sites places spatial constr aints on allowable residues in that interface.