title: 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 Zn(2+) ions bind sequentially, with the first Zn(2+) binding to a CysHis3 site with a nanomolar binding affinity, and the second Zn(2+) 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 Zn(2+) coordination. Our data supports a model in which the intracellular M3M4 domain senses high cytosolic Zn(2+) concentrations and regulates the plasma membrane levels of the hZIP4 transporter in response to Zn(2+) binding.
authors.affiliation: Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA 01609, USA. rdempski@wpi.edu.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Bafaro
authors.forename: Sagar
authors.initials: S
authors.lastname: Antala
authors.forename: Tuong-Vi
authors.initials: TV
authors.lastname: Nguyen
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Dzul
authors.forename: Brian
authors.initials: B
authors.lastname: Doyon
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Stemmler
authors.forename: Robert E
authors.initials: RE
authors.lastname: Dempski
mesh_terms: D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000737:chemistry* / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D027682:Cation Transport Proteins; D064267:Intrinsically Disordered Proteins; C486791:SLC39A4 protein, human; D006639:Histidine; D015032:Zinc; D003545:Cysteine