Premium
The Binuclear Transport Center of ZupT: A Zinc Transporter from E. coli
Author(s) -
Roberts Cameron,
Mitra Bharati
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.08910
Subject(s) - transporter , chemistry , zinc , transport protein , biochemistry , membrane transport , atp binding cassette transporter , membrane , gene , organic chemistry
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 Glu 123 (orange). His 148 and Glu 152 round out site 1 (red) and Asn 120 , Asn 149 , and Glu 181 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 His 119 (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.