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

Abstract

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.

Rational engineering of an elevator-type metal transporter ZIP8 reveals a conditional selectivity filter critically involved in determining substrate specificity

“…Our initial focus was put on the Zn/Cd selectivity because the two metals, both in the IIB column of the periodic table, share similar properties in coordination chemistry. Indeed, the previous studies have indicated that Zn 2+ and Cd 2+ use the same transport pathway through multi-metal ZIPs 32 , 44 , and distinguishing them requires a delicate but less understood mechanism. To precisely measure small kinetic isotope effect of an enzyme, a commonly used approach is the internal competition assay in which a mixture of two competing substrates labeled with light and heavy isotopes respectively is applied to the enzyme for processing 45 .…”

“…More importantly, the mutagenesis and functional studies conducted in this work unraveled key molecular determinants of substrate specificity of ZIP8, in particular a conditional selectivity filter. Among the four residues substituted in the 4M variant, only E343 participates in metal binding at the M1 site which is the primary transport site of the ZIPs 6,44,46,57 . Previous multiple sequence analysis has predicted E343 as a key residue involved in determining substrate specificity 14,15 , but its role was evidenced only when the transport dead E343H variant was rescued by the Q180H mutation in the 2M variant (Fig.…”

Utilizing a divalent metal ion transporter to control biogenic nanoparticle synthesis

“…ZupT is suspected to transport many divalent cations, as many ZIP family proteins (ZRT, IRT-like Proteins) have been shown to have a broad substrate range (Guerinot, 2000 ). The metal-binding sites of ZupT interacted similarity with zinc and cadmium (Roberts et al., 2021 ), and ZupT was directly shown to enable uptake of cobalt, cadmium, and manganese by E. coli (Grass et al., 2005 ; Taudte & Grass, 2010 ).…”

Structural insights into the elevator-type transport mechanism of a bacterial ZIP metal transporter

“…While the kinetic studies using the cell-based transport assays suggested a carrier mode for eukaryotic and prokaryotic ZIPs 35 – 42 , a bacterial ZIP from Bordetella bronchiseptica (ZIPB or BbZIP) reconstituted in proteoliposome was reported to behave like an ion channel 43 . The studies on other bacterial ZIPs reconstituted in proteoliposome, however, showed Michaelis-Menten type kinetics 44 , 45 . Structural studies have provided critical insights into the transport mechanisms for numerous membrane transport proteins.…”