Cryo-EM structure of a eukaryotic zinc transporter at a low pH suggests its Zn²⁺-releasing mechanism

Abstract

Zinc transporter 8 (ZnT8) is mainly expressed in pancreatic islet β cells and is responsible for H+-coupled uptake (antiport) of Zn2+ into insulin secretory granules. Structures of human ZnT8 and its prokaryotic homolog YiiP have provided structural basis for constructing a plausible transport cycle for Zn2+. However, the mechanistic role that H+ plays in the transport process remains elusive. Here we present two cryo-EM structures of ZnT8 from Xenopus tropicalis (xtZnT8) captured in the presence of either abundant Zn2+ or abundant H+. Combined with a microscale thermophoresis analysis, our data suggest that binding of Zn2+ to the transmembrane Zn2+-binding site drives xtZnT8 to the outward-facing state. Surprisingly, binding of H+ to xtZnT8 is not sufficient to drive the transporter to an inward-facing state, suggesting that protonation alone is not a determining factor to establish an inward-facing conformation during Zn2+ transport. Instead, the role of protonation appears to unbind and release Zn2+ from the transmembrane site in the outward-facing state of xtZnT8, thus allowing an inward-facing isomerization to occur for the next cycle.