Unzipping the ZIPs: Transport Mechanism and Substrate Specificity of ZIP Metal Transporters

Living organisms have evolved sophisticated systems to exploit unique properties of d-block metals for catalysis, cell signaling, and gene regulation. A challenge is how to efficiently and specifically uptake, excrete, and distribute/redistribute these low-abundance trace elements at the systemic and cellular levels. Transition metal transporters are central players in these processes by controlling the flux of metals across cell and organelle membranes. Since discovered in 1990s, the Zrt-/Irt-like protein (ZIP) family has been known for its pivotal roles in maintaining homeostasis of zinc, iron and manganese¹. Human ZIPs are broadly involved in physiological and pathological processes, some of which are promising cancer drug targets. In this talk, I will present our recent studies on two interrelated topics: transport mechanism and substrate specificity. My group solved the first ZIP structure, revealing a new fold of membrane transporter with a binuclear metal center sitting at the transport site². The recent apo state structure uncovered a rigid body rotation of a 4-transmembrane helix (TM) bundle relative to the other TMs. The computationally generated and biochemically validated outward-facing conformation model revealed a vertical motion of the 4-TM bundle, which exclusively carries metal substrate, by approximately 8 Å toward the extracellular side against the other static TMs. Together with the results of evolutionary covariance analysis, mutagenesis and functional studies, these findings allowed us to conclude that the ZIPs utilize the elevator-type transport mechanism to achieve alternating access³. The ZIPs may exhibit distinct substrate specificity even within the same subgroup. To identify the determinants of substrate specificity, we applied an integrated approach to rationally alter the substrate preference of a multi-metal transporter ZIP8⁴. By systematically replacing the differentially conserved residues with the counterparts in zinc-preferring ZIP4, we created a zinc-specific quadruple variant 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. These research provide critical insights into the working mechanism of the ZIPs.

References

1. Hu, J., Toward unzipping the ZIP metal transporters: structure, evolution, and implications on drug discovery against cancer. FEBS J 2021, 288 (20), 5805-5825.
2. Zhang, T.;  Liu, J.;  Fellner, M.;  Zhang, C.;  Sui, D.; Hu, J., Crystal structures of a ZIP zinc transporter reveal a binuclear metal center in the transport pathway. Sci Adv 2017, 3 (8), e1700344.
3. Zhang, Y.;  Jiang, Y.;  Gao, K.;  Sui, D.;  Yu, P.;  Su, M.;  Wei, G. W.; Hu, J., Structural insights into the elevator-type transport mechanism of a bacterial ZIP metal transporter. Nat Commun 2023, 14 (1), 385.
4. Jiang, Y.;  Li, Z.;  Sui, D.;  Sharma, G.;  Wang, T.;  MacRenaris, K.;  Takahashi, H.;  Merz, K. M.; Hu, J., Rational engineering of an elevator-type metal transporter ZIP8 reveals a conditional selective filter critically involved in determining substrate specificity. (under revision) 2023.