Cd2+ and the N-terminal metal-binding domain protect the putative membranous CPC motif of the Cd2+-ATPase of Listeria monocytogenes

CadA, the Cd²⁺-ATPase of Listeria monocytogenes, contains four cysteine residues: two in the CTNC (Cys-Thr-Asn-Cys) sequence in the cytoplasmic metal-binding domain (MBD), and two in the CPC (Cys-Pro-Cys) sequence in the membrane domain. Taking advantage of ΔMBD, a truncated version of CadA that lacks the MBD but which still acts as a functional Cd²⁺-ATPase [Bal, Mintz, Guillain and Catty (2001) FEBS Lett. 506, 249—252], we analysed the role of the membrane cysteine residues (studied using ΔMBD) separately from that of the cysteine residues of the MBD, which were studied using full-length CadA. The role of the cysteines was assessed by reacting ΔMBD and CadA with N-ethylmaleimide (NEM), an SH-specific reagent, in the presence or absence of Cd²⁺. We show here that (i) in both ΔMBD and CadA, the cysteine residues in the CPC motif are essential for phosphorylation; (ii) in both proteins, Cd²⁺ protects against alkylation by NEM; and (iii) in the absence of Cd²⁺, the MBD of CadA also protects against alkylation by NEM. Our results suggest that the CPC motif is present in the membrane Cd²⁺ transport site(s) and that the MBD protects these site(s).