The cadmium transport sites of CadA, the Cd2+-ATPase from Listeria monocytogenes

Abstract

CadA, the Cd2+-ATPase from Listeria monocytogenes, belongs to the Zn2+/Cd2+/Pb2+-ATPase bacterial subfamily of P1B-ATPases that ensure detoxification of the bacteria. Whereas it is the major determinant of Listeria resistance to Cd2+, CadA expressed in Saccharomyces cerevisiae severely decreases yeast tolerance to Cd2+ (Wu, C. C., Bal, N., Pérard, J., Lowe, J., Boscheron, C., Mintz, E., and Catty, P. (2004) Biochem. Biophys. Res. Commun. 324, 1034-1040). This phenotype, which reflects in vivo Cd2+-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 Cys354 and Cys356 in TM6 as well as Asp692 in TM8 and Met149 in TM3 could participate at the Cd2+-binding site(s). In the canonical Cys-Pro-Cys motif of P1B-ATPases, the two cysteines act at distinct steps in the transport mechanism, Cys356 being directly involved in Cd 2+ binding, while Cys356 seems to be required for Cd 2+ occlusion. This confirms an earlier observation that the two equivalent Cys of Ccc2, the yeast Cu+-ATPase, also act at different steps. In TM4, Glu164, which is conserved among P1B- ATPases, may be required for Cd2+ release. Finally, analysis of the role of Cd2+ in the phosphorylation from ATP and from Pi of the mutants suggests that two Cd2+ ions are involved in the reaction cycle of CadA. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.