The Cadmium Transport Sites of CadA, the Cd2+-ATPase from Listeria monocytogenes

Chen-Chou Wu,Aurélie Gardarin,Anne Martel,Elisabeth Mintz,Florent Guillain,Patrice Catty

doi:10.1074/jbc.m604658200

Abstract

CadA, the Cd(2+)-ATPase from Listeria monocytogenes, belongs to the Zn(2+)/Cd(2+)/Pb(2+)-ATPase bacterial subfamily of P(1B)-ATPases that ensure detoxification of the bacteria. Whereas it is the major determinant of Listeria resistance to Cd(2+), CadA expressed in Saccharomyces cerevisiae severely decreases yeast tolerance to Cd(2+) (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 Cd(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(354) and Cys(356) in TM6 as well as Asp(692) in TM8 and Met(149) in TM3 could participate at the Cd(2+)-binding site(s). In the canonical Cys-Pro-Cys motif of P(1B)-ATPases, the two cysteines act at distinct steps in the transport mechanism, Cys(354) being directly involved in Cd(2+) binding, while Cys(356) 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, Glu(164), which is conserved among P(1B)-ATPases, may be required for Cd(2+) release. Finally, analysis of the role of Cd(2+) in the phosphorylation from ATP and from P(i) of the mutants suggests that two Cd(2+) ions are involved in the reaction cycle of CadA.

Highlights

P-type ATPase is the generic name for a class of membrane proteins that pump cations against their electrochemical gradient at the expense of ATP
P-type ATPases possess in their membranous domain one or more sites which bind the cation(s) to be transported. These so-called transport sites have been mainly studied on Ca2ϩ, Naϩ/Kϩ, and Hϩ/Kϩ-ATPases (P2 subfamily) and Hϩ-ATPases (P3 subfamily). It emerged from these studies that whatever the ionic selectivity of the pump, the transport site(s) involved at least three transmembrane helices, including those two that are directly linked to the catalytic loop
In SERCA1a, the Ca2ϩ-transport sites consist of Glu, Asp, Asn, and Thr, which bind Ca2ϩ through their side chain [10], plus some hydrophobic amino acids, which participate through their backbone carbonyl group, and a water molecule

Summary

Introduction

The present work aims to identify the amino acids of the CadA transmembrane domain that are involved in Cd2ϩ binding and release during the transport cycle. This value results from two effects of Cd2ϩ, which on the one hand inhibits the endogenous ATPase activity, as illustrated in the membrane fraction containing the non-functional D398A mutant, and on the other hand activates CadA (Ref. 31 and Fig. 4).

Results

Conclusion