Zinc binding alters the conformational dynamics and drives the transport cycle of the cation diffusion facilitator yiip

YiiP is a secondary transporter that couples Zn²⁺ transport to the proton motive force. Structural studies of YiiP from prokaryotes and Znt8 from humans have revealed three different Zn²⁺ sites and a conserved homodimeric architecture. These structures define the inward-facing and outward-facing states that characterize the archetypal alternating access mechanism of transport. To study the effects of Zn²⁺ binding on the conformational transition, we use cryo-EM together with molecular dynamics simulation to compare structures of YiiP from Shewanella oneidensis in the presence and absence of Zn²⁺. To enable single-particle cryo-EM, we used a phage-display library to develop a Fab antibody fragment with high affinity for YiiP, thus producing a YiiP/Fab complex. To perform MD simulations, we developed a nonbonded dummy model for Zn²⁺ and validated its performance with known Zn²⁺-binding proteins. Using these tools, we find that, in the presence of Zn²⁺, YiiP adopts an inward-facing conformation consistent with that previously seen in tubular crystals. After removal of Zn²⁺ with high-affinity chelators, YiiP exhibits enhanced flexibility and adopts a novel conformation that appears to be intermediate between inward-facing and outward-facing states. This conformation involves closure of a hydrophobic gate that has been postulated to control access to the primary transport site. Comparison of several independent cryo-EM maps suggests that the transition from the inward-facing state is controlled by occupancy of a secondary Zn²⁺ site at the cytoplasmic membrane interface. This work enhances our understanding of individual Zn²⁺ binding sites and their role in the conformational dynamics that govern the transport cycle.