Role of Individual Zinc Binding Sites in the Cation Diffusion Facilitator YIIP

Abstract

YiiP is a dimeric, zinc/proton bacterial antiporter that belongs to the family of Cation Diffusion Facilitators. YiiP contributes to zinc homeostasis by exporting Zn(II) from the cytosol to the periplasm. YiiP has three Zn(II) binding sites: site a in the membrane domain, site B in an intracellular loop between transmembrane helices M2 and M3, and site C harboring two ions at the dimer interface between the C-terminal domains (CTD) of the homodimer. Here we present high-resolution cryoEM structures of YiiP in complex with a Fab antibody fragment in the presence and absence of Zn(II). Comparison of these structures reveals a dramatic conformational change that affects the accessibility of the transport sites. This conformational change includes bending of M2 and M5 helices, disordering of the M2-M3 loop, and a ∼25° kink between the CTD and the membrane domain. These results are consistent with Molecular Dynamics simulations that show how the M2-M3 loop becomes flexible without the stabilizing effect of Zn(II) bound in site B. To further characterize the role of individual Zn(II) sites in these structural changes, single-site mutations were introduced at each site: D51A at site A, D70A at site B, and D287A at site C. All mutations reduced the stoichiometry of Zn(II) binding, measured by atomic absorption spectroscopy, and abolished Zn(II) transport. Cryo-EM structures of the three mutants were obtained from detergent solubilized YiiP-Fab complexes that were fully loaded with Zn(II). Structures of D51A and D287A mutants revealed conformations comparable to the holo state. In contrast, the D70A mutant produced a structure that resembled the apo state. These findings suggest that binding of Zn(II) to site B induces a conformational change that allows Zn(II) to bind to the transport sites in advance of the transport process.