Metal selectivity of a Cd-, Co-, and Zn-transporting P_1B-type ATPase

The P_1B-ATPases, a family of transmembrane metal transporters important for transition metal homeostasis in all organisms, are subdivided into classes based on sequence conservation and metal specificity. The multifunctional P_1B-4-ATPase CzcP is part of the cobalt, zinc, and cadmium resistance system from the metal-tolerant, model organism Cupriavidus metallidurans. Previous work revealed the presence of an unusual soluble metal-binding domain (MBD) at the CzcP N-terminus, but the nature, extent, and selectivity of the transmembrane metal-binding site (MBS) of CzcP have not been resolved. Using homology modeling, we show that four wholly conserved amino acids from the transmembrane (TM) domain (Met²⁵⁴, Ser⁴⁷⁴, Cys⁴⁷⁶, and His⁸⁰⁷) are logical candidates for the TM MBS, which may communicate with the MBD via interactions with the first TM helix. Metal-binding analyses indicate that wild-type (WT) CzcP has three MBSs, and data on N-terminally truncated (ΔMBD) CzcP suggest the presence of a single TM MBS. Electronic absorption and electron paramagnetic resonance spectroscopic analyses of ΔMBD CzcP and variant proteins thereof provide insight into the details of Co²⁺ coordination by the TM MBS. These spectroscopic data, combined with in vitro functional studies of WT and variant CzcP proteins, show that the side chains of Met²⁵⁴, Cys⁴⁷⁶, and His⁸⁰⁷ contribute to Cd²⁺, Co²⁺, and Zn²⁺ binding and transport, whereas the side chain of Ser⁴⁷⁴ appears to play a minimal role. By comparison to other P_1B-4-ATPases, we suggest that an evolutionarily adapted flexibility in the TM region likely afforded CzcP the ability to transport Cd²⁺ and Zn²⁺ in addition to Co²⁺.