The role of zinc binding sites in the cation diffusion facilitator YiiP.

Abstract

YiiP is a zinc/proton antiporter that couples zinc export to the proton motive force. YiiP binds zinc at three sites: site A in the transmembrane domain, site B in a cytoplasmic loop between transmembrane helices M2 and M3, and site C in the cytoplasmic, C-terminal domain (CTD). Previous Cryo-EM analysis revealed that global removal of zinc by EDTA produced a novel inward occluded state that was postulated to be an intermediate between the inward and outward facing states. In this study we introduced mutations to study binding affinities and conformational effects of each individual site. Cryo-EM analysis showed that disruption of site A produces little change, but that disruption of site B induces the inward occluded state observed previously. Double mutation at site C produced nonnative oligomers, indicating that site C is important for assembly of the native homodimer. Molecular dynamic simulations with zinc removed from site B showed increased mobility of the CTD and disordering of the M2M3 loop in agreement with the structural studies. Binding affinity was quantified by microscale thermophoresis using either citrate or NTA to buffer the concentration of free zinc. The results demonstrate nanomolar affinity at sites A and C and micromolar affinity at site B. Site A is highly sensitive to pH changes with Kd increasing from 1 nM to 302 μM as pH decreases from 7.4 to 5.6. Based on these observations, we propose a transport mechanism whereby zinc is initially recruited at site B and that transfer to site A induces conformational changes leading to transport. The pH dependence of site A is key to energy coupling by ensuring high affinity binding from the cytoplasm and then promoting release to the more acidic periplasmic milieau.