The Cadmium Transport Sites of CadA, the Cd super(2+)-ATPase from Listeria monocytogenes

CadA, the Cd super(2+)-ATPase from Listeria monocytogenes, belongs to the Zn super(2+)/Cd super(2+)/Pb super(2+)-ATPase bacterial subfamily of P sub(1B)-ATPases that ensure detoxification of the bacteria. Whereas it is the major determinant of Listeria resistance to Cd super(2+), CadA expressed in Saccharomyces cerevisiae severely decreases yeast tolerance to Cd super(2+) (Wu, C. C., Bal, N., Perard, J., Lowe, J., Boscheron, C., Mintz, E., and Catty, P. (2004) BIOCHEM: Biophys. Res. Commun. 324, 1034-1040). This phenotype, which reflects in vivo Cd super(2+)-transport activity, was used to select from 33 point mutations, shared out among the eight transmembrane (TM) segments of CadA, those that affect the activity of the protein. Six mutations affecting CadA were found: M149A in TM3; E164A in TM4; C354A, P355A, and C356A in TM6; and D692A in TM8. Functional studies of the six mutants produced in Sf9 cells revealed that Cys super(354) and Cys super(356) in TM6 as well as Asp super(692) in TM8 and Met super(149) in TM3 could participate at the Cd super(2+)-binding site(s). In the canonical Cys-Pro-Cys motif of P sub(1B)-ATPases, the two cysteines act at distinct steps in the transport mechanism, Cys super(354) being directly involved in Cd super(2+) binding, while Cys super(356) seems to be required for Cd super(2+) occlusion. This confirms an earlier observation that the two equivalent Cys of Ccc2, the yeast Cu super(+)-ATPase, also act at different steps. In TM4, Glu super(164), which is conserved among P sub(1B)-ATPases, may be required for Cd super(2+) release. Finally, analysis of the role of Cd super(2+) in the phosphorylation from ATP and from P sub(i) of the mutants suggests that two Cd super(2+) ions are involved in the reaction cycle of CadA.