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

An integrated experimental and computational approach reveals an ion selectivity filter in multi-metal transporter ZIP8 that is rationally engineered to increase preference toward Zn2+ over Cd2+. Engineering of transporters to alter substrate specificity as desired holds great potential for applications, including metabolic engineering. However, the lack of knowledge on molecular mechanisms of substrate specificity hinders designing effective strategies for transporter engineering. Here, we applied an integrated approach to rationally alter the substrate preference of ZIP8, a Zrt-/Irt-like protein (ZIP) metal transporter with multiple natural substrates, and uncovered the determinants of substrate specificity. By systematically replacing the differentially conserved residues with the counterparts in the zinc transporter ZIP4, we created a zinc-preferring quadruple variant (Q180H/E343H/C310A/N357H), which exhibited largely reduced transport activities towards Cd2+, Fe2+, and Mn2+ whereas increased activity toward Zn2+. Combined mutagenesis, modeling, covariance analysis, and computational studies revealed a conditional selectivity filter which functions only when the transporter adopts the outward-facing conformation. The demonstrated approach for transporter engineering and the gained knowledge about substrate specificity will facilitate engineering and mechanistic studies of other transporters.

Automated summary

An integrated experimental and computational approach was used to study the substrate selectivity of the multi-metal transporter ZIP8. By rationally engineering the transporter, a zinc-preferring variant was created, which showed reduced transport activities towards other metals and increased activity towards zinc. The study revealed a conditional selectivity filter in the transporter and provided knowledge about substrate specificity, which can be applied to other transporter engineering and mechanistic studies.