The Zinc and Iron Binuclear Transport Center of ZupT, a ZIP Transporter from Escherichia coli.

ZupT fromEscherichia coliis a member of the Zrt-/Irt-like Protein (ZIP) transporter family, which is responsible for zinc uptake during zinc-sufficient conditions. ZIP transporters have been shown to transport different divalent metal ions including zinc, iron, manganese, and cadmium. In this study, we show that ZupT has an asymmetric binuclear metal center in the transmembrane domain; one metal-binding site, M1, binds zinc, cadmium, and iron, while the other, M2, binds iron only and with higher affinity than M1. Using site-specific mutagenesis and transport activity measurements in whole cells and proteoliposomes, we show that zinc is transported from M1, while iron is transported from M2. The two sites share a common bridging ligand, a conserved glutamate residue. M1 and M2 have ligands from highly conserved motifs in transmembrane domains 4 and 5. Additionally, M2 has a ligand from transmembrane domain 6, a glutamate residue, which is conserved in the gufA subfamily of ZIP transporters, including ZupT and the human ZIP11. Unlike cadmium, iron transport from M2 does not inhibit the zinc transport activity but slightly stimulates it. This stimulation of activity is mediated through the bridging carboxylate ligand. The binuclear zinc-iron binding center in ZupT has likely evolved to enable the transport of essential metals from two different sites without competition; a similar mechanism of metal transport is likely to be found in the gufA subfamily of ZIP transporter proteins.

Structural Role of the First Four Transmembrane Helices in ZntA, a P1B-Type ATPase from Escherichia coli.

ZntA from Escherichia coli confers resistance to toxic concentrations of Pb2+, Zn2+, and Cd2+. It is a member of the P1B-ATPase transporter superfamily, which includes the human Cu+-transporting proteins ATP7A and ATP7B. P1B-type ATPases typically have a hydrophilic N-terminal metal-binding domain and eight transmembrane helices. A splice variant of ATP7B was reported, which has 100-fold higher night-specific expression in the pineal gland; it lacks the entire N-terminal domain and the first four transmembrane helices. Here, we report our findings with Δ231-ZntA, a similar truncation we created in ZntA. Δ231-ZntA has no in vivo and greatly reduced in vitro activity. It binds one metal ion per dimer at the transmembrane site, with a 15-19000-fold higher binding affinity, indicating highly significant changes in the dimer structure of Δ231-ZntA relative to that of ZntA. Cd2+ has the highest affinity for Δ231-ZntA, in contrast to ZntA, which has the highest affinity for Pb2+. Site-specific mutagenesis of the metal-binding residues, 392Cys, 394Cys, and 714Asp, showed that there is considerable flexibility at the metal-binding site, with any two of these three residues able to bind Zn2+ and Pb2+ unlike in ZntA. However, Cd2+ binds to only 392Cys and 714Asp, with 394Cys not involved in Cd2+ binding. Three-dimensional homology models show that there is a dramatic difference between the ZntA and Δ231-ZntA dimer structures, which help to explain these observations. Therefore, the first four transmembrane helices in ZntA and P1B-type ATPases play an important role in maintaining the correct dimer structure.