Zinc Binding Induces Conformational Changes in the Large Intracellular Loop of the Human Zinc Transporter hZIP4

Abstract

The human (h)ZIP4 transporter is a plasma membrane protein, primarily expressed in intestinal cells, that functions to increase the cytosolic concentration of zinc. While first identified for its role in the zinc deficiency disease acrodermatitis enteropathica, hZIP4 has been implicated in pancreatic, liver and brain cancers where its surface expression is abnormally increased. Surface expression of hZIP4 is regulated by post-translational modifications in response to zinc concentration. At high zinc concentrations, hZIP4 undergoes ubiquitination and proteasomal degradation. hZIP ubiquitination likely occurs at a conserved lysine within a large intracellular loop located between transmembrane domains 3 and 4 (M3M4) and requires a histidine-rich domain located on the M3M4 loop. Based on these observations, the hZIP4 intracellular loop has been hypothesized to act as a zinc sensor. We have recombinantly expressed and purified the large intracellular M3M4 loop of hZIP4, as well as mutants in the histidine-rich domain, for metal binding and structural studies. The large intracellular loop coordinates two zinc ions sequentially, with the first zinc ion binding with nanomolar affinity to a CysHis3 site and the second zinc ion binding to a His4 site with a lower binding affinity. Structural analysis using circular dichroism revealed that the intracellular loop is an intrinsically disordered region, and it remains disordered upon zinc binding. Our data support a model in which structural changes in the large intracellular loop of hZIP4 in response to zinc regulate the plasma membrane levels of the hZIP4 transporter.