Zinc transporter mutations linked to acrodermatitis enteropathica disrupt function and cause mistrafficking
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100269
ISSN1083-351X
AutoresEziz Kuliyev, Chi Zhang, Dexin Sui, Jian Hu,
Tópico(s)RNA regulation and disease
ResumoZIP4 is a representative member of the Zrt-/Irt-like protein (ZIP) transporter family and responsible for zinc uptake from diet. Loss-of-function mutations of human ZIP4 (hZIP4) drastically reduce zinc absorption, causing a life-threatening autosomal recessive disorder, acrodermatitis enteropathica (AE). These mutations occur not only in the conserved transmembrane zinc transport machinery, but also in the extracellular domain (ECD) of hZIP4, which is only present in a fraction of mammalian ZIPs. How these AE-causing ECD mutations lead to ZIP4 malfunction has not be fully clarified. In this work, we characterized all seven confirmed AE-causing missense mutations in hZIP4-ECD and found that the variants exhibited completely abolished zinc transport activity in a cell-based transport assay. Although the variants were able to be expressed in HEK293T cells, they failed to traffic to the cell surface and were largely retained in the ER with immature glycosylation. When the corresponding mutations were introduced in the ECD of ZIP4 from Pteropus Alecto, a close homolog of hZIP4, the variants exhibited structural defects or reduced thermal stability, which likely accounts for intracellular mistrafficking of the AE-associated variants and as such a total loss of zinc uptake activity. This work provides a molecular pathogenic mechanism for AE. ZIP4 is a representative member of the Zrt-/Irt-like protein (ZIP) transporter family and responsible for zinc uptake from diet. Loss-of-function mutations of human ZIP4 (hZIP4) drastically reduce zinc absorption, causing a life-threatening autosomal recessive disorder, acrodermatitis enteropathica (AE). These mutations occur not only in the conserved transmembrane zinc transport machinery, but also in the extracellular domain (ECD) of hZIP4, which is only present in a fraction of mammalian ZIPs. How these AE-causing ECD mutations lead to ZIP4 malfunction has not be fully clarified. In this work, we characterized all seven confirmed AE-causing missense mutations in hZIP4-ECD and found that the variants exhibited completely abolished zinc transport activity in a cell-based transport assay. Although the variants were able to be expressed in HEK293T cells, they failed to traffic to the cell surface and were largely retained in the ER with immature glycosylation. When the corresponding mutations were introduced in the ECD of ZIP4 from Pteropus Alecto, a close homolog of hZIP4, the variants exhibited structural defects or reduced thermal stability, which likely accounts for intracellular mistrafficking of the AE-associated variants and as such a total loss of zinc uptake activity. This work provides a molecular pathogenic mechanism for AE. Zinc is an essential micronutrient for any living organism. As the second most abundant transition metal element in humans after iron, zinc is required for function of over 2000 transcription factors and activity of approximately 300 enzymes (1McCall K.A. Huang C. Fierke C.A. Function and mechanism of zinc metalloenzymes.J. Nutr. 2000; 130: 1437S-1446SCrossref PubMed Google Scholar). It has been estimated that zinc ions bind to nearly 3000 proteins, which account for ∼10% of the proteins encoded in human genome (2Andreini C. Banci L. Bertini I. Rosato A. Counting the zinc-proteins encoded in the human genome.J. Proteome Res. 2006; 5: 196-201Crossref PubMed Scopus (602) Google Scholar). Studies have shown that zinc deficiency may affect many biological processes, causing growth retardation, immune dysfunction, diarrhea, delayed sexual maturation, and skin lesions (3Maret W. Sandstead H.H. Zinc requirements and the risks and benefits of zinc supplementation.J. Trace Elem. Med. Biol. 2006; 20: 3-18Crossref PubMed Scopus (627) Google Scholar). 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Dufner-Beattie J. Petris M.J. Andrews G. Eide D.J. Acrodermatitis enteropathica mutations affect transport activity, localization and zinc-responsive trafficking of the mouse ZIP4 zinc transporter.Hum. Mol. Genet. 2004; 13: 563-571Crossref PubMed Scopus (116) Google Scholar, 8Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. The acrodermatitis enteropathica gene ZIP4 encodes a tissue-specific, zinc-regulated zinc transporter in mice.J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 9Dufner-Beattie J. Weaver B.P. Geiser J. Bilgen M. Larson M. Xu W. Andrews G.K. The mouse acrodermatitis enteropathica gene Slc39a4 (Zip4) is essential for early development and heterozygosity causes hypersensitivity to zinc deficiency.Hum. Mol. Genet. 2007; 16: 1391-1399Crossref PubMed Scopus (96) Google Scholar, 10Geiser J. Venken K.J. De Lisle R.C. Andrews G.K. A mouse model of acrodermatitis enteropathica: Loss of intestine zinc transporter ZIP4 (Slc39a4) disrupts the stem cell niche and intestine integrity.PLoS Genet. 2012; 8e1002766Crossref PubMed Scopus (81) Google Scholar). AE is fatal without treatment, but lifelong high-dose zinc supplementation on daily basis can effectively alleviate the symptoms (11Perafan-Riveros C. Franca L.F. Alves A.C. Sanches Jr., J.A. Acrodermatitis enteropathica: Case report and review of the literature.Pediatr. Dermatol. 2002; 19: 426-431Crossref PubMed Scopus (112) Google Scholar), implying the presence of secondary low-affinity zinc absorption mechanism(s). ZIP4 is a representative member of the Zrt-/Irt-like protein (ZIP) family (solute carrier 39A, SLC39A) consisting of divalent transition metal transporters ubiquitous in all the kingdoms of life (12Kambe T. Yamaguchi-Iwai Y. Sasaki R. Nagao M. 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Physiological roles of zinc transporters: Molecular and genetic importance in zinc homeostasis.J. Physiol. Sci. 2017; 67: 283-301Crossref PubMed Scopus (153) Google Scholar, 18Jeong J. Eide D.J. The SLC39 family of zinc transporters.Mol. Aspects Med. 2013; 34: 612-619Crossref PubMed Scopus (242) Google Scholar). ZIP4 is specifically expressed on the apical side of enterocytes in the small intestine and also in the kidney where it is believed to be involved in zinc reabsorption from urine (18Jeong J. Eide D.J. The SLC39 family of zinc transporters.Mol. Aspects Med. 2013; 34: 612-619Crossref PubMed Scopus (242) Google Scholar). Topologically, ZIP4 has a transmembrane domain (TMD), which is generally conserved in the entire ZIP family, and a large extracellular domain (ECD), which is only present in a fraction of the family members (19Zhang T. Sui D. Hu J. Structural insights of ZIP4 extracellular domain critical for optimal zinc transport.Nat. Commun. 2016; 7: 11979Crossref PubMed Scopus (37) Google Scholar, 20Hu J. Wille H. Schmitt-Ulms G. The evolutionary unZIPping of a dimerization motif—a Comparison of ZIP and PrP architectures.Pathogens. 2018; 7: 4Crossref Scopus (3) Google Scholar). The AE-causing mutations are evenly distributed along the 12 exons of zip4 gene without showing hotspot (21Schmitt S. Kury S. Giraud M. Dreno B. Kharfi M. Bezieau S. An update on mutations of the SLC39A4 gene in acrodermatitis enteropathica.Hum. Mutat. 2009; 30: 926-933Crossref PubMed Scopus (95) Google Scholar, 22Kury S. Kharfi M. Schmitt S. Bezieau S. Clinical utility gene card for: Acrodermatitis enteropathica.Eur. J. Hum. Genet. 2012; 20Google Scholar). As a result, half of the missense AE-causing mutations are mapped in the ECD and the other half in the TMD where the zinc transport machinery is located. A previous work has investigated several corresponding mutations in the TMD of mouse ZIP4 (mZIP4) but only one mutation in the ECD (equivalent to the P200L mutation in human ZIP4, hZIP4) was studied in the same report (7Wang F. Kim B.E. Dufner-Beattie J. Petris M.J. Andrews G. Eide D.J. Acrodermatitis enteropathica mutations affect transport activity, localization and zinc-responsive trafficking of the mouse ZIP4 zinc transporter.Hum. Mol. Genet. 2004; 13: 563-571Crossref PubMed Scopus (116) Google Scholar). The P200L variant of hZIP4 was also characterized in a recent report (23Hoch E. Levy M. Hershfinkel M. Sekler I. Elucidating the H(+) coupled Zn(2+) transport mechanism of ZIP4; implications in acrodermatitis enteropathica.Int. J. Mol. Sci. 2020; 21Crossref Scopus (4) Google Scholar). So far, it is still unclear about how the other AE-causing mutations in the ECD affect hZIP4 function and the corresponding molecular mechanisms are also unknown. The crystal structure of the ECD of ZIP4 from Pteropus Alecto (black fruit bat, pZIP4-ECD), which shares 68% sequence identity with hZIP4-ECD, provides a structural framework to deduce structural impacts of the AE-causing mutations on this accessory domain required for optimal zinc transport (19Zhang T. Sui D. Hu J. Structural insights of ZIP4 extracellular domain critical for optimal zinc transport.Nat. Commun. 2016; 7: 11979Crossref PubMed Scopus (37) Google Scholar). In this work, we functionally characterized all the seven confirmed AE-associated variants in a human cell line (HEK293T) and biophysically studied the purified variants of pZIP4-ECD harboring the corresponding mutations. We found that all the variants showed little zinc transport activity in the cell-based transport assay. Although the variants were expressed, the cell surface expression was barely detectable. The variants were found to be immaturely glycosylated and aberrantly retained in the ER. For the purified pZIP4-ECD variants, biophysical studies revealed that the mutations caused structural defects or reduced thermal stability, providing a possible causative explanation for ZIP4 mistrafficking and dysfunction. A total of 17 missense mutations in hZIP4-ECD have been documented, of which seven are confirmed AE-causing mutations and the others are benign single-nucleotide polymorphisms (SNPs), according to the MASTERMIND database (https://mastermind.genomenon.com/) and an early review (21Schmitt S. Kury S. Giraud M. Dreno B. Kharfi M. Bezieau S. An update on mutations of the SLC39A4 gene in acrodermatitis enteropathica.Hum. Mutat. 2009; 30: 926-933Crossref PubMed Scopus (95) Google Scholar). Mapping these residues on the hZIP4-ECD structural model (generated using SWISS-MODEL (24Waterhouse A. Bertoni M. Bienert S. Studer G. Tauriello G. Gumienny R. Heer F.T. de Beer T.A.P. Rempfer C. Bordoli L. Lepore R. Schwede T. SWISS-MODEL: Homology modelling of protein structures and complexes.Nucleic Acids Res. 2018; 46: W296-W303Crossref PubMed Scopus (2937) Google Scholar)) shows that the residues subjected to AE-causing mutations (C62R, R95C, A99T, N106K, P200L, Q303H, and C309Y) are all in the structured regions, whereas most of the residues where the SNPs occur are in loops or disordered regions (Fig. 1). In this work, we characterized all the seven AE-causing mutations, including four homozygous mutations (C62R, P200L, Q303H, and C309Y) (5Kury S. Dreno B. Bezieau S. Giraudet S. Kharfi M. Kamoun R. Moisan J.P. Identification of SLC39A4, a gene involved in acrodermatitis enteropathica.Nat. Genet. 2002; 31: 239-240Crossref PubMed Scopus (384) Google Scholar, 6Wang K. Zhou B. Kuo Y.M. Zemansky J. Gitschier J. A novel member of a zinc transporter family is defective in acrodermatitis enteropathica.Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 25Kury S. Kharfi M. Kamoun R. Taieb A. Mallet E. Baudon J.J. Glastre C. Michel B. Sebag F. Brooks D. Schuster V. Scoul C. Dreno B. Bezieau S. Moisan J.P. Mutation spectrum of human SLC39A4 in a panel of patients with acrodermatitis enteropathica.Hum. Mutat. 2003; 22: 337-338Crossref PubMed Scopus (52) Google Scholar, 26Nakano A. Nakano H. Nomura K. Toyomaki Y. Hanada K. Novel SLC39A4 mutations in acrodermatitis enteropathica.J. Invest. Dermatol. 2003; 120: 963-966Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and three heterozygous mutations (R95C, A99T, and N106K) (6Wang K. Zhou B. Kuo Y.M. Zemansky J. Gitschier J. A novel member of a zinc transporter family is defective in acrodermatitis enteropathica.Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 26Nakano A. Nakano H. Nomura K. Toyomaki Y. Hanada K. Novel SLC39A4 mutations in acrodermatitis enteropathica.J. Invest. Dermatol. 2003; 120: 963-966Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 27Jung A.G. Mathony U.A. Behre B. Kury S. Schmitt S. Zouboulis C.C. Lippert U. Acrodermatitis enteropathica: An uncommon differential diagnosis in childhood - first description of a new sequence variant.J. Dtsch. Dermatol. Ges. 2011; 9: 999-1002PubMed Google Scholar), and studied their impacts on hZIP4 function. The hZIP4 variants with a C-terminal HA tag were transiently expressed in HEK293T cells and applied to the cell-based zinc transport assay (6Wang K. Zhou B. Kuo Y.M. Zemansky J. Gitschier J. A novel member of a zinc transporter family is defective in acrodermatitis enteropathica.Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 7Wang F. Kim B.E. Dufner-Beattie J. Petris M.J. Andrews G. Eide D.J. Acrodermatitis enteropathica mutations affect transport activity, localization and zinc-responsive trafficking of the mouse ZIP4 zinc transporter.Hum. Mol. Genet. 2004; 13: 563-571Crossref PubMed Scopus (116) Google Scholar, 28Zhang T. Kuliyev E. Sui D. Hu J. The histidine-rich loop in the extracellular domain of ZIP4 binds zinc and plays a role in zinc transport.Biochem. J. 2019; 476: 1791-1803Crossref PubMed Scopus (9) Google Scholar, 29Zhang T. Sui D. Zhang C. Cole L. Hu J. Asymmetric functions of a binuclear metal center within the transport pathway of a human zinc transporter ZIP4.FASEB J. 2020; 34: 237-247Crossref PubMed Scopus (7) Google Scholar). As shown in Figure 2A, in sharp contrast to the wild-type hZIP4, none of the cells expressing any variants uptook more zinc than the blank group in which the cells were transfected with an empty vector, which means that the variants have no detectable zinc transport activity with 10 μM of Zn2+ in culture media. For the wild-type hZIP4, the previous studies have shown that zinc transport activity reaches plateau at 10 μM of Zn2+ under the same condition (28Zhang T. Kuliyev E. Sui D. Hu J. The histidine-rich loop in the extracellular domain of ZIP4 binds zinc and plays a role in zinc transport.Biochem. J. 2019; 476: 1791-1803Crossref PubMed Scopus (9) Google Scholar, 29Zhang T. Sui D. Zhang C. Cole L. Hu J. Asymmetric functions of a binuclear metal center within the transport pathway of a human zinc transporter ZIP4.FASEB J. 2020; 34: 237-247Crossref PubMed Scopus (7) Google Scholar). To examine whether any activity can be detected at higher zinc concentration, the P200L variant was tested at various zinc concentrations up to 50 μM, but the results only confirmed no detectable activity (Fig. S1).Figure 2Expression and functional characterization of hZIP4 and the variants. A, cell-based zinc transport assay of the AE-associated variants with 10 μM Zn2+ in the medium. The data points of one representative experiment out of 2–6 independent experiments are shown as black dots, and the means and the standard deviations are indicated by bars. B, total expression hZIP4 and the variants detected by western blot using anti-HA antibody. β-actin was used as loading control. C, processing of hZIP4 by PNGase F and Endo H glycosidases. D, zinc transport assay of the N261Q variant. Data are expressed as average ± standard deviation (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The previous study on mZIP4 has shown that the AE-causing mutations in the TMD led to significantly reduced expression levels, likely due to accelerated degradation (7Wang F. Kim B.E. Dufner-Beattie J. Petris M.J. Andrews G. Eide D.J. Acrodermatitis enteropathica mutations affect transport activity, localization and zinc-responsive trafficking of the mouse ZIP4 zinc transporter.Hum. Mol. Genet. 2004; 13: 563-571Crossref PubMed Scopus (116) Google Scholar). To examine whether the AE-causing mutations in the ECD have similar effects, expression of the variants was detected in the whole-cell samples using an anti-HA antibody in western blot. As shown in Figure 2B, all the variants were expressed, but the wild-type hZIP4 has two discrete bands at approximately 75 kDa, whereas the AE-associated variants have only one band corresponding to the lower band of the wild-type protein. When treated with PNGase F, an enzyme cleaving the glycosidic bond directly linked with the asparagine residue, the two bands of the wild-type protein merged into a single one with a smaller apparent molecular weight, indicating that the expressed hZIP4 harbors two distinct glycans (7Wang F. Kim B.E. Dufner-Beattie J. Petris M.J. Andrews G. Eide D.J. Acrodermatitis enteropathica mutations affect transport activity, localization and zinc-responsive trafficking of the mouse ZIP4 zinc transporter.Hum. Mol. Genet. 2004; 13: 563-571Crossref PubMed Scopus (116) Google Scholar). When treated with Endo H, which is a glycosidase selectively hydrolyzing high-mannose or some hybrid glycans, the lower band was processed, whereas the upper band was resistant (Fig. 2C), indicating that the species in the upper band has a complex glycan, whereas the lower band represents a species with immature glycosylation. In contrast, the P200L variant was completely processed by Endo H, and the resulting species is of the same apparent molecular weight as the one corresponding to the lower band of the processed wild-type protein. Thus, although the total expression levels of the variants were not drastically affected by the mutations in the ECD, defects in glycosylation were observed for all the AE-associated variants. Proteins expressed at cell surface are often modified by N-glycosylation, which may play a key role in protein function (30Spiro R.G. Protein glycosylation: Nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds.Glycobiology. 2002; 12: 43R-56RCrossref PubMed Scopus (973) Google Scholar). As the AE-associated variants losing zinc transport activity are concomitant with defects in glycosylation, we asked whether the lack in glycosylation is responsible for activity loss. Using the NetNGlyc server (http://www.cbs.dtu.dk/services/NetNGlyc/), we identified a potential glycosylation site at N261 in an "NxS/T" motif. We then generated the N261Q variant and expressed it in HEK293T cells. In western blot, the N261Q variant showed a single band with an apparent molecular weight smaller than the lower band of the wild-type protein (Fig. 2B), confirming that N261 is indeed the only N-glycosylation site in hZIP4. Importantly, zinc transport assay of the N261Q variant only showed a modest reduction of transport activity (Fig. 2D), indicating that glycosylation at N261 is not pivotal for zinc transport and therefore lack of glycosylation of the AE-associated variants does not account for loss of zinc transport activity. As the glycans are added to the client membrane proteins in the ER, processed in the ER and then in the Golgi in a stepwise manner (31Marinko J.T. Huang H. Penn W.D. Capra J.A. Schlebach J.P. Sanders C.R. Folding and misfolding of human membrane proteins in health and disease: From single molecules to cellular proteostasis.Chem. Rev. 2019; 119: 5537-5606Crossref PubMed Scopus (62) Google Scholar), immature glycosylation is an indicator of defect in intracellular trafficking. To examine potential mislocalization, we tested cell surface expression levels of the AE-associated variants by applying the anti-HA antibody to the nonpermeabilized cells fixed with formaldehyde (7Wang F. Kim B.E. Dufner-Beattie J. Petris M.J. Andrews G. Eide D.J. Acrodermatitis enteropathica mutations affect transport activity, localization and zinc-responsive trafficking of the mouse ZIP4 zinc transporter.Hum. Mol. Genet. 2004; 13: 563-571Crossref PubMed Scopus (116) Google Scholar, 19Zhang T. Sui D. Hu J. Structural insights of ZIP4 extracellular domain critical for optimal zinc transport.Nat. Commun. 2016; 7: 11979Crossref PubMed Scopus (37) Google Scholar, 32Mao X. Kim B.E. Wang F. Eide D.J. Petris M.J. A histidine-rich cluster mediates the ubiquitination and degradation of the human zinc transporter, hZIP4, and protects against zinc cytotoxicity.J. Biol. Chem. 2007; 282: 6992-7000Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 33Zhang C. Sui D. Zhang T. Hu J. Molecular basis of zinc-dependent endocytosis of human ZIP4 transceptor.Cell Rep. 2020; 31: 107582Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). After extensive wash, nonspecific bound antibody was removed, leaving those specifically bound with the C-terminal HA tag at cell surface. As shown in Figure 3A, all the AE-associated variants had substantially reduced cell surface levels when compared with the wild-type hZIP4 in western blot. Consistent with the zinc transport data, the N261Q variant missing the N-linked glycan had significantly higher level of cell surface expression than the variants linked with disease, indicating that the N-glycosylation at N261 is not a key factor for hZIP4 surface expression. We then applied immunofluorescence imaging to locate the AE-associated variants expressed in cells. To do that, the cells transiently expressing the wild type or the variants were fixed and permeabilized with formaldehyde and TX-100, followed by staining with an FITC-labeled anti-HA antibody. The samples were then checked under confocal laser scanning microscope (CLSM). Consistent with the western blot shown in Figure 2B, the wild type and the variants were expressed in HEK293T cells at comparable levels (Fig. 3B). To detect the transporters expressed at cell surface, the cells were fixed with formaldehyde and treated with the same anti-HA antibody, followed by extensive washing and checking under CLSM. In Figure 3B, fluorescence signals at cell surface can be convincingly detected for the wild-type hZIP4 and the N261Q variant, whereas the signals of the AE-associated variants imaged under the same conditions were not detectable. Collectively, our data strongly indicate that the cell surface expression levels of the AE-associated variants were largely suppressed, which is likely responsible for loss of zinc transport activity. Since all the AE-associated variants were expressed but not presented at cell surface, we then asked where the variants are located in cells. Endo H treatment experiment suggests that the variants are likely trapped in the ER (Fig. 2C). To test this, we examined colocalization of hZIP4 and its variants with an ER-resident protein, calreticulin, and quantified the results using Pearson's correlation coefficient (PCC). As shown in Figure 4, the staining of the wild-type hZIP4-HA (red) was partially superimposed with those of calreticulin (green), resulting in a yellow/orange color. The calculated PCC (0.37 ± 0.06) indicates that a significant portion of hZIP4 expressed in HEK293T cells were retained in the ER, which is consistent with the results of the Endo H treatment, which showed that more than half of the expressed hZIP4 were not maturely glycosylated. For all the AE-associated variants, more overlapped signals were detected and the PCCs (0.52–0.66) are significantly higher than that of the wild-type protein (p < 0.01) (Fig. 4), indicating that these variants were retained in the ER to a greater extent than the wild-type protein. Next, we attempted to clarify the molecular basis of the variants' mistrafficking. Given the difficulty of obtaining adequate amount of purified full-length hZIP4 or hZIP4-ECD for biophysical characterization, we turned to the isolated pZIP4-ECD, which we have previously crystallized and biochemically characterized (19Zhang T. Sui D. Hu J. Structural insights of ZIP4 extracellular domain critical for optimal zinc transport.Nat. Commun. 2016; 7: 11979Crossref PubMed Scopus (37) Google Scholar, 28Zhang T. Kuliyev E. Sui D. Hu J. The histidine-rich loop in the extracellular domain of ZIP4 binds zinc and plays a role in zinc transport.Biochem. J. 2019; 476: 1791-1803Crossref PubMed Scopus (9) Google Scholar). Given the high sequence identity, we believe that the results obtained from the study of purified pZIP4-ECD would help to understand how the disease-causing mutations affect the biophysical properties of the human counterpart. We introduced mutations in pZIP4-ECD on the residues topologically equivalent to those in hZIP4 (C64R, R87C, A91T, D98K, P193L, Q299H, and C305Y). The variants were expressed and purified in the same way as the wild-type protein, but the final yields were lower. Particularly, the yields of two variants (D98K and C305Y) were too low to allow for further characterization. Therefore, we focused on the other five variants in later study. In size-exclusion chromatography, all the variants were eluted as a single peak with apparent molecular weights at or greater than that of a homodimer (66 kDa), except that the Q299H variant has a smaller apparent molecular weight at approximately 50 kDa (Fig. 5). Consistently, the hydrodynamic diameter of the Q299H variant (7.4 ± 1.2 nm), which was determined by dynamic light scattering, is significantly smaller than that of the wild-type protein (10.6 ± 0.3 nm, p < 0.05). As only one species was detected in both the size-exclusion chromatography and the dynamic light scatter experiment, the monomer–dimer equilibrium appears to strongly favor one side, but the current data cannot tell whether this variant preserves dimerization or becomes monomeric in solution. The purified proteins were applied to circular dichroism (CD) spectrometer, and the data were analyzed on the K2D3 server to estimate secondary structure contents (34Louis-Jeune C. Andrade-Navarro M.A. Perez-Iratxeta C. Prediction of protein secondary structure from circular dichroism using theoretically derived spectra.Proteins. 2012; 80: 374-381Crossref PubMed Scopus (354) Google Scholar). As shown in Figure 6, the CD spectrum of the wild-type pZIP4-ECD has a high α-helical content (66 ± 2%) with the characteristic minima at 208 nm and 222 nm and maximum at 195 nm, which is consistent with the reported crystal structure (19Zhang T. Sui D. Hu J. Structural insights of ZIP4 extracellular domain critical for optimal zinc transport.Nat. Commun. 2016; 7: 11979Crossref PubMed Scopus (37) Google Scholar). When compared with the wild-type protein, three variants exhibited lower levels of α-helical content – R87C (56 ± 3%), A91T (59 ± 3%), and Q299H (56 ± 2%) (p < 0.05). In addition, the ratios of ellipticities (θ) at 222 nm and 208 nm were significantly reduced. The θ222/θ208 ratio is an indicator of whether α-helices are packed to form coiled-coil-like tertiary structures (35Zhou N.E. Kay C.M. Hodges R.S. Synthetic model proteins. Positional effects of interchain hydrophobic interactions on stability of two-stranded alpha-helical coiled-coils.J. Biol. 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