Concomitant disorder and high‐affinity zinc binding in the human zinc‐ and iron‐regulated transport protein 4 intracellular loop

Abstract

The human zinc-and iron-regulated transport protein 4 (hZIP4) protein is the major plasma membrane protein responsible for the uptake of zinc in the body, and as such it plays a key role in cellular zinc homeostasis. hZIP4 plasma membrane levels are regulated through post-translational modification of its large, disordered, histidine-rich cytosolic loop (ICL2) in response to intracellular zinc concentrations. Here, structural characteristics of the isolated disordered loop region, both in the absence and presence of zinc, were investigated using nuclear magnetic resonance (NMR) spectroscopy. NMR chemical shifts, coupling constants and temperature coefficients of the apoprotein, are consistent with a random coil with minor propensities for transient polyproline Type II helices and β-strand in regions implicated in post-translational modifications. The ICL2 protein remains disordered upon zinc binding, which induces exchange broadening. Paramagnetic relaxation enhancement experiments reveal that the histidine-rich region in the apoprotein makes transient tertiary contacts with predicted post-translational modification sites. The residue-specific data presented here strengthen the relationship between hZIP4 post-translational modifications, which impact its role in cellular zinc homeostasis, and zinc sensing by the intracellular loop.Broader Impact Statement: Intrinsically disordered proteins often undergo coupled binding-folding in order to perform their biological functions. We find that the intracellular loop region of the zinc transporter human zinc-and iron-regulated transport protein 4 (hZIP4) remains disordered upon zinc binding, despite having a high affinity for zinc. As the intracellular loop regulates post-translational modification of hZIP4 in a zinc-dependent manner, the absence of a pronounced conformational change suggests an alternative mechanism in which changes in surface charge distribution upon zinc binding influence post-translational modification. Published by WileyFurthermore, the zinc sensing mechanism employed by the ICL2 protein demonstrates that highaffinity interactions can occur in the presence of conformational disorder.

The two-domain elevator-type mechanism of zinc-transporting ZIP proteins

“…Mechanistically, it is hence possible that the N terminus of nine-TM ZIPs structurally and functionally replaces the distinctly longer and histidine-rich ICL2 of the human members. Noteworthy, all human ZIPs are predicted to share an eight-TM topology, and hence, they lack an intracellular tail, but the ICL2 instead contributes to zinc binding, thereby regulating Zn 2+ transport activity ( 42 , 43 ).…”

Molecular Basis of Zinc-Dependent Endocytosis of Human ZIP4 Transceptor

“…The second conserved histidine-rich segment (region IV) is on the second cytosolic loop connecting TM3 and TM4 (referred to as L2), which is a characteristic feature of many ZIP family members ( Jeong and Eide, 2013 ). It has been shown that the metal chelating residues in the L2 bind zinc with nanomolar affinity ( Bafaro et al, 2015 , 2019 ) and are required for zinc-induced hZIP4 degradation ( Mao et al, 2007 ). However, diminishing zinc binding capability of the L2 by replacing all five histidine residues (H438, H441, H443, H446, and H448) with glycine residues did not affect either endocytosis or zinc sensing ( Figure 2E ), which is consistent with the result of alanine substitution of these histidine residues in hZIP4 ( Mao et al, 2007 ).…”

“…However, diminishing zinc binding capability of the L2 by replacing all five histidine residues (H438, H441, H443, H446, and H448) with glycine residues did not affect either endocytosis or zinc sensing ( Figure 2E ), which is consistent with the result of alanine substitution of these histidine residues in hZIP4 ( Mao et al, 2007 ). Because C436 has been indicated to solely confer nanomolar affinity toward zinc ion for the isolated L2 peptide ( Bafaro et al, 2015 , 2019 ), we generated the C436A variant and the C436A/H438G/H441G/H443G/H446G/ H448G variant for which zinc binding at the L2 is supposed to be greatly abrogated and completely eliminated, respectively. As shown in Figures 2F and 2G , no defect in zinc sensing can be detected for these two variants.…”

Toward unzipping the ZIP metal transporters: structure, evolution, and implications on drug discovery against cancer

“…The IL2 of HsZIP4 has been extensively studied. The isolated IL2 peptide of HsZIP4 is unstructured as revealed in an NMR study [61] and the highly dynamic nature of the IL2 was also manifested by partial proteolysis of purified full length HsZIP4 [62]. The histidine residues in the IL2 bind zinc ions with nanomolar affinity, and zinc titration in the NMR study showed that, although the IL2 binds two zinc ions, there is no defined manner of zinc‐binding, suggesting that the zinc‐binding sites are highly dynamic and undergoing a fast equilibrium among different conformers [61].…”

“…The isolated IL2 peptide of HsZIP4 is unstructured as revealed in an NMR study [61] and the highly dynamic nature of the IL2 was also manifested by partial proteolysis of purified full length HsZIP4 [62]. The histidine residues in the IL2 bind zinc ions with nanomolar affinity, and zinc titration in the NMR study showed that, although the IL2 binds two zinc ions, there is no defined manner of zinc‐binding, suggesting that the zinc‐binding sites are highly dynamic and undergoing a fast equilibrium among different conformers [61]. These results support the notion that the IL2 functions as a zinc sensor which has been shown to be essential for zinc‐induced endocytic degradation when cells are exposed to tens to hundreds of micromolar of extracellular zinc ions.…”