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.

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.

ZupT is a member of the ZIP family of transporters which are responsible for increasing the intracellular concentration of zinc from E. coli to humans. Defects in these transporters are linked to zinc deficiency, cancer, and cardiovascular disease. The mechanism of transport in this family is still not well‐understood, partly due to the bottleneck in the expression and purification of these integral membrane proteins. Recently a structure was solved of a ZIP transporter from B. bronchiseptica. The structure revealed a cadmium binuclear metal transport site, which is relatively uncommon for metal transporters. Using the crystal structure, a homology model was built for ZupT which shares 32% identity and a conserved binuclear center. In the ZupT model the metal ions are separated by roughly 4.2 A and coordinated by a bidentate Glu123 (orange). His148 and Glu152 round out site 1 (red) and Asn120, Asn149, and Glu181 site 2 (yellow). In this work, solution studies including competitive metal titrations were carried out with purified ZupT. Our results show that ZupT binds one equivalent of zinc, its primary substrate, at physiological concentrations. In contrast, it binds two equivalents of iron, a secondary substrate. Site directed mutagenesis was carried out to determine which residues are important in coordinating zinc and iron. Zinc and iron both bind to a ‘primary’ transport site (site 1‐red), while iron also binds to a secondary site (site 2‐yellow). Activity assays were used to determine the relationship between binding and function. ZupT showed transport activity with both iron and zinc; additionally, it was observed that iron positively regulated zinc activity. Our results also indicated that the highly conserved residue His119 (blue) is not essential for binding of either zinc or iron, but is important for overall transport activity, probably by facilitating metal release. Overall these results show that ZIP transporters have a single zinc transport site which can be regulated by iron binding to an adjacent secondary site. Binuclear metal center of ZupT. Utilizing the crystal structure of ZIPB from B. bronchiseptica a homology model was made with the SWISS‐MODEL server. Visualization of the binculear center was carried out with UCSF CHIMERA.