The large intracellular loop of hZIP4 is an intrinsically disordered zinc binding domain

Abstract

The human (h) ZIP4 transporter is a plasma membrane protein which functions to increase the cytosolic concentration of zinc. hZIP4 transports zinc into intestinal cells and therefore has a central role in the absorption of dietary zinc. hZIP4 has eight transmembrane domains and encodes a large intracellular loop between transmembrane domains III and IV, M3M4. Previously, it has been postulated that this domain regulates hZIP4 levels in the plasma membrane in a zinc-dependent manner. The objective of this research was to examine the zinc binding properties of the large intracellular loop of hZIP4. Therefore, we have recombinantly expressed and purified M3M4 and showed that this domain binds two zinc ions. Using a combination of site-directed mutagenesis, metal binding affinity assays, and X-ray absorption spectroscopy, we demonstrated that the two Zn²⁺ ions bind sequentially, with the first Zn²⁺ binding to a CysHis₃ site with a nanomolar binding affinity, and the second Zn²⁺ binding to a His₄ site with a weaker affinity. Circular dichroism spectroscopy revealed that the M3M4 domain is intrinsically disordered, with only a small structural change induced upon Zn²⁺ coordination. Our data supports a model in which the intracellular M3M4 domain senses high cytosolic Zn²⁺ concentrations and regulates the plasma membrane levels of the hZIP4 transporter in response to Zn²⁺ binding.