Targeting the selectivity filter to drastically alter the activity and substrate spectrum of a promiscuous metal transporter

d-Block metal transporters are attractive engineering targets for selectively enriching or excluding metals in living organisms. However, systematic efforts to engineer these transporters have been hindered by limited knowledge on the determinants of substrate specificity. Here, we applied a focused-screen approach to human ZIP8, a promiscuous d-block divalent metal transporter, by systematically changing the residues that form a proposed selectivity filter at the entrance of the transport pathway. Library screening using a cell-based transport assay quantified by ICP-MS led to the identification of the variants with drastically altered transport activities and substrate preferences, including a variant that exhibits new activities for the non-substrate metals VO²⁺ and Cu²⁺. Together with the identification of Pb²⁺ as a new substrate of ZIP8, these findings indicate that the ZIP fold is highly adaptable and amenable for transporting a wide range of metals, making it a promising scaffold to generate novel metal transporters for applications.