Mechanism of Ph-dependent Zn2+ Binding in the Zinc Transporter Protein YiiP

Transition metal ions such as Zn2+, Mn2+, Co2+, and Fe2+ play crucial roles in organisms from all kingdoms of life. The homeostasis of these ions is mainly regulated by a group of secondary transporters from the cation diffusion facilitator (CDF) family. The mammalian zinc transporters (ZnTs), a subfamily of CDF, have been an important target for study as they are associated with several diseases, such as diabetes, delayed growth and osteopenia, Alzheimer’s disease, and Parkinsonism. The bacterial homolog of ZnTs, YiiP, is the first CDF transporter with a determined structure and is used as a model for studying the structural and mechanistic properties of CDF transporters. On the other hand, Molecular dynamics simulation has emerged as a valuable computational tool for exploring the physical basis of biological macromolecules' structure and function with atomic precision at femtosecond resolution. This work aims to elucidate the roles of the three Zn$2+ binding sites found on each YiiP protomer and the role of protons in the transport process of CDFs, which remain under debate despite previous thermodynamic and structural studies on YiiP. Cryo-EM, microscale thermophoresis (MST) and molecular dynamics (MD) simulations were used to address these questions. With a Zn2+ model that accurately reproduces experimental structures of the binding clusters, the dynamical influence of zinc binding on the transporter was accessed through MD simulations, which was consistent with the new cryo-EM structures. Zinc binding affinities obtained through MST were used to infer the stoichiometry of Zn2+/H+ antiport in combination with a microscopic thermodynamic model and constant pH simulations. The most likely microstates of H$^+$ and Zn2+ binding indicated a transport stoichiometry of 1 Zn2+ to 2-3 H+ depending on the external pH. A model describing the entire transport cycle of YiiP was finally built on these findings, providing insight into the structural and mechanistic properties of CDF transporters.