Symmetry analysis and ancestral sequence reconstruction reveal a symmetrical translocation pathway and activity determinants of ZIP metal transporters

Abstract Membrane transporters frequently exhibit internal structural symmetry, reflecting evolutionary origins through gene duplication and fusion events, and this feature has been widely used to infer transport mechanisms. Yet its application to the Zrt‐/Irt‐like protein (ZIP) family of divalent metal transporters, which are crucial for trace metal homeostasis, has not been fully explored. Here, we apply combined symmetry analysis and ancestral sequence reconstruction (ASR) to study the ZIP family. We show that internal symmetry is broadly present across prokaryotic ZIPs and is more prominent in reconstructed ancestral sequences, supporting an evolutionary pathway in which the modern 8‐transmembrane (TM) ZIP fold arose from duplication, circular permutation, and fusion of an ancestral 4‐TM protein. Leveraging this symmetry framework, we identify a continuous and symmetric metal translocation pathway composed of symmetric entry, transport, and exit sites in the ZIP fold and define gates that ensure alternating access. Application of this structural model to human ZIP4 enables identification of the gate‐forming residues, and functional studies reveal that two residues (T529 and V533) in the external gate play a crucial role in controlling metal transport. In addition, comparison with ancestral sequences uncovers a set of LIV‐1 subfamily‐specific metal‐chelating residues (D504, E541, and D544) that break the symmetry in the ancestral sequences. Functional studies showed that these residues play distinct roles in transport. Together, our study demonstrates that combining internal symmetry analysis and ASR for the ZIP family facilitates elucidation of the metal translocation mechanism and identification of subfamily‐specific features that regulate transport function.