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• W2923141331 abstract "The transport and ion-coupling mechanisms of ZIP transporters remain largely uncharacterized. Previous work in our laboratory has revealed that the solute carrier family 39 member A2 (SLC39A2/ZIP2) increases its substrate transport rate in the presence of extracellular H+. Here, we used a combination of in silico and in vitro techniques involving structural modeling, mutagenesis, and functional characterization in HEK293 cells to identify amino acid residues potentially relevant for both the ZIP2–H+ interaction and substrate binding. Our ZIP2 models revealed a cluster of charged residues close to the substrate–translocation pore. Interestingly, the H63A substitution completely abrogated pH sensitivity, and substitutions of Glu-67 and Phe-269 altered the pH and voltage modulation of transport. In contrast, substitution of Glu-106, which might be part of a dimerization interface, altered pH but not voltage modulation. Substitution of Phe-269, located close to the substrate-binding site, also affected substrate selectivity. These findings were supported by an additional model of ZIP2 that was based on the structure of a prokaryotic homolog, Bordetella bronchiseptica ZrT/Irt-like protein (bbZIP), and in silico pKa calculations. We also found that residues Glu-179, His-175, His-202, and Glu-276 are directly involved in the coordination of the substrate metal ion. We noted that, unlike bbZIP, human ZIP2 is predicted to harbor a single divalent metal-binding site, with the charged side chain of Lys-203 replacing the second bound ion. Our results provide the first structural evidence for the previously observed pH and voltage modulation of ZIP2-mediated metal transport, identify the substrate-binding site, and suggest a structure-based transport mechanism for the ZIP2 transporter. The transport and ion-coupling mechanisms of ZIP transporters remain largely uncharacterized. Previous work in our laboratory has revealed that the solute carrier family 39 member A2 (SLC39A2/ZIP2) increases its substrate transport rate in the presence of extracellular H+. Here, we used a combination of in silico and in vitro techniques involving structural modeling, mutagenesis, and functional characterization in HEK293 cells to identify amino acid residues potentially relevant for both the ZIP2–H+ interaction and substrate binding. Our ZIP2 models revealed a cluster of charged residues close to the substrate–translocation pore. Interestingly, the H63A substitution completely abrogated pH sensitivity, and substitutions of Glu-67 and Phe-269 altered the pH and voltage modulation of transport. In contrast, substitution of Glu-106, which might be part of a dimerization interface, altered pH but not voltage modulation. Substitution of Phe-269, located close to the substrate-binding site, also affected substrate selectivity. These findings were supported by an additional model of ZIP2 that was based on the structure of a prokaryotic homolog, Bordetella bronchiseptica ZrT/Irt-like protein (bbZIP), and in silico pKa calculations. We also found that residues Glu-179, His-175, His-202, and Glu-276 are directly involved in the coordination of the substrate metal ion. We noted that, unlike bbZIP, human ZIP2 is predicted to harbor a single divalent metal-binding site, with the charged side chain of Lys-203 replacing the second bound ion. Our results provide the first structural evidence for the previously observed pH and voltage modulation of ZIP2-mediated metal transport, identify the substrate-binding site, and suggest a structure-based transport mechanism for the ZIP2 transporter. Correction: Unraveling the structural elements of pH sensitivity and substrate binding in the human zinc transporter SLC39A2 (ZIP2).Journal of Biological ChemistryVol. 295Issue 13PreviewVOLUME 294 (2019) PAGES 8046–8063 Full-Text PDF Open Access Zinc (Zn2+) is a transition metal required in mammalian cells as a cofactor and structural component of a wide variety of cellular proteins, as well as a signaling molecule. Consequently, intracellular Zn2+ bioavailability is essential for various cellular components and signaling pathways necessary for the survival and growth of mammalian cells. To ensure an adequate control of intracellular Zn2+ levels, cells possess specific transport proteins on both plasma and organellar membranes as well as specialized storage and carrier proteins in the cytoplasm (1Bafaro E. Liu Y. Xu Y. Dempski R.E. The emerging role of zinc transporters in cellular homeostasis and cancer.Signal Transduct. Target Ther. 2017; 2 (29218234)1702910.1038/sigtrans.2017.29Crossref PubMed Scopus (138) Google Scholar). Zn2+ transporter proteins are grouped into two solute carrier families, SLC39 and SLC30, that differ in terms of the direction of metal ion transport. SLC39 transporters accumulate Zn2+ into the cytoplasm (i.e. import from the extracellular medium and export from intracellular organelles), whereas SLC30 transporters mobilize Zn2+ out of the cytoplasm in the opposite direction (2Baltaci A.K. Yuce K. Zinc transporter proteins.Neurochem. Res. 2018; 43 (29243032): 517-53010.1007/s11064-017-2454-yCrossref PubMed Scopus (54) Google Scholar). The SLC39 family, also known as ZIP (Zrt-, Irt-like proteins), is constituted by 14 different members, which are subdivided into subfamilies I, II, LIV-1, and gufA, according to their sequence similarities (3Kambe T. Tsuji T. Hashimoto A. Itsumura N. The physiological, biochemical, and molecular roles of zinc transporters in zinc homeostasis and metabolism.Physiol. Rev. 2015; 95 (26084690): 749-78410.1152/physrev.00035.2014Crossref PubMed Scopus (561) Google Scholar). To date, there is no direct structural information available for any full-length mammalian ZIP transporter. However, all the ZIP members are predicted to have eight transmembrane domains, with their N and C termini facing the extracellular space (1Bafaro E. Liu Y. Xu Y. Dempski R.E. The emerging role of zinc transporters in cellular homeostasis and cancer.Signal Transduct. Target Ther. 2017; 2 (29218234)1702910.1038/sigtrans.2017.29Crossref PubMed Scopus (138) Google Scholar, 4Jeong J. Eide D.J. The SLC39 family of zinc transporters.Mol. Aspects Med. 2013; 34 (23506894): 612-61910.1016/j.mam.2012.05.011Crossref PubMed Scopus (279) Google Scholar). Interestingly, recent computational studies using co-evolution contact predictions and Rosetta ab initio structure prediction generated the first molecular model for a mammalian ZIP transporter (5Antala S. Ovchinnikov S. Kamisetty H. Baker D. Dempski R.E. Computation and functional studies provide a model for the structure of the zinc transporter hZIP4.J. Biol. Chem. 2015; 290 (25971965): 17796-1780510.1074/jbc.M114.617613Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). This model of human ZIP4, a member of the subfamily LIV-1, exhibits eight transmembrane helices (TMH), 6The abbreviations used are: TMHtransmembrane helixbbZIPB. bronchiseptica ZrT/Irt-like proteinHRPhorseradish peroxidaseDMEMDulbecco's modified Eagle's mediumAUCarea under the curve. in which TMHs 2, 4, 5, and 7 form a helical bundle containing the putative metal-coordination site. Alanine replacement of the histidine and aspartic acid residues present in the proposed metal-coordination site altered the Zn2+ transport kinetics, validating this structural model (5Antala S. Ovchinnikov S. Kamisetty H. Baker D. Dempski R.E. Computation and functional studies provide a model for the structure of the zinc transporter hZIP4.J. Biol. Chem. 2015; 290 (25971965): 17796-1780510.1074/jbc.M114.617613Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The described metal-coordination site resembles that of the crystal structure of the SLC30 family homolog YiiP (6Lu M. Fu D. Structure of the zinc transporter YiiP.Science. 2007; 317 (17717154): 1746-174810.1126/science.1143748Crossref PubMed Scopus (297) Google Scholar) (i.e. Escherichia coli Zn2+/H+ antiporter), suggesting a common structural element for the different Zn2+ transporters. Another interesting aspect revealed by these studies is that the predicted contacts used as a basis to build the model could only be observed when ZIP4 was modeled as a dimer in which the TMHs form the dimer interface (1Bafaro E. Liu Y. Xu Y. Dempski R.E. The emerging role of zinc transporters in cellular homeostasis and cancer.Signal Transduct. Target Ther. 2017; 2 (29218234)1702910.1038/sigtrans.2017.29Crossref PubMed Scopus (138) Google Scholar, 5Antala S. Ovchinnikov S. Kamisetty H. Baker D. Dempski R.E. Computation and functional studies provide a model for the structure of the zinc transporter hZIP4.J. Biol. Chem. 2015; 290 (25971965): 17796-1780510.1074/jbc.M114.617613Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). This finding is supported by previous evidence of homodimerization of other ZIP transporters, such as ZIP2, ZIP7, and ZIP13, or the heteromer of ZIP6 and ZIP10 (1Bafaro E. Liu Y. Xu Y. Dempski R.E. The emerging role of zinc transporters in cellular homeostasis and cancer.Signal Transduct. Target Ther. 2017; 2 (29218234)1702910.1038/sigtrans.2017.29Crossref PubMed Scopus (138) Google Scholar). Although not included in the computational model, ZIP transporters are predicted to possess a large cytoplasmic loop between TMHs 3 and 4, which contains a variety of zinc-coordination sites likely functioning as a regulatory region that senses and responds to changes in intracellular Zn2+ concentration (1Bafaro E. Liu Y. Xu Y. Dempski R.E. The emerging role of zinc transporters in cellular homeostasis and cancer.Signal Transduct. Target Ther. 2017; 2 (29218234)1702910.1038/sigtrans.2017.29Crossref PubMed Scopus (138) Google Scholar). Recently, the first experimental evidence of the overall structure emerged through the crystallization of a prokaryotic ZIP homolog, bbZIP, showing remarkable overall agreement with the previously described theoretical model (7Zhang T. Liu J. Fellner M. Zhang C. Sui D. Hu J. Crystal structures of a ZIP zinc transporter reveal a binuclear metal center in the transport pathway.Sci. Adv. 2017; 3 (28875161)e170034410.1126/sciadv.1700344Crossref PubMed Scopus (95) Google Scholar). Based on the X-ray structure, a structural model of the human ZIP4 protein was derived, and several structural elements of the protein, such as the substrate-binding site, have been identified (7Zhang T. Liu J. Fellner M. Zhang C. Sui D. Hu J. Crystal structures of a ZIP zinc transporter reveal a binuclear metal center in the transport pathway.Sci. Adv. 2017; 3 (28875161)e170034410.1126/sciadv.1700344Crossref PubMed Scopus (95) Google Scholar). transmembrane helix B. bronchiseptica ZrT/Irt-like protein horseradish peroxidase Dulbecco's modified Eagle's medium area under the curve. In terms of the functional properties of the SLC39/ZIP family, members of this family transport a wide variety of divalent metals in addition to Zn2+, including iron (Fe2+), manganese (Mn2+), copper (Cu2+), and cadmium (Cd2+) (1Bafaro E. Liu Y. Xu Y. Dempski R.E. The emerging role of zinc transporters in cellular homeostasis and cancer.Signal Transduct. Target Ther. 2017; 2 (29218234)1702910.1038/sigtrans.2017.29Crossref PubMed Scopus (138) Google Scholar). Even though the transport mechanism of ZIPs is not yet fully understood, it has been reported that the transport process is not dependent on ATP hydrolysis (8Gaither L.A. Eide D.J. Functional expression of the human hZIP2 zinc transporter.J. Biol. Chem. 2000; 275 (10681536): 5560-556410.1074/jbc.275.8.5560Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 9Gaither L.A. Eide D.J. The human ZIP1 transporter mediates zinc uptake in human K562 erythroleukemia cells.J. Biol. Chem. 2001; 276 (11301334): 22258-2226410.1074/jbc.M101772200Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) and, for some of the ZIP transporters such as ZIP2, ZIP8, and ZIP14, a Zn2+/HCO3− symport mechanism has been proposed (8Gaither L.A. Eide D.J. Functional expression of the human hZIP2 zinc transporter.J. Biol. Chem. 2000; 275 (10681536): 5560-556410.1074/jbc.275.8.5560Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 10He L. Girijashanker K. Dalton T.P. Reed J. Li H. Soleimani M. Nebert D.W. ZIP8, member of the solute-carrier-39 (SLC39) metal-transporter family: characterization of transporter properties.Mol. Pharmacol. 2006; 70 (16638970): 171-18010.1124/mol.106.024521Crossref PubMed Scopus (267) Google Scholar, 11Girijashanker K. He L. Soleimani M. Reed J.M. Li H. Liu Z. Wang B. Dalton T.P. Nebert D.W. Slc39a14 gene encodes ZIP14, a metal/bicarbonate symporter: similarities to the ZIP8 transporter.Mol. Pharmacol. 2008; 73 (18270315): 1413-142310.1124/mol.107.043588Crossref PubMed Scopus (256) Google Scholar). In contrast, studies conducted with a bacterial ZIP transporter suggested that this protein might function as an ion-selective channel in which Zn2+ is incorporated into the cytoplasm via an electro-diffusion mechanism (12Lin W. Chai J. Love J. Fu D. Selective electrodiffusion of zinc ions in a Zrt-, Irt-like protein, ZIPB.J. Biol. Chem. 2010; 285 (20876577): 39013-3902010.1074/jbc.M110.180620Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). SLC39A2, also known as ZIP2, belongs to the ZIP subfamily II, and it was cloned and functionally characterized for the first time by Gaither and Eide in the year 2000 (8Gaither L.A. Eide D.J. Functional expression of the human hZIP2 zinc transporter.J. Biol. Chem. 2000; 275 (10681536): 5560-556410.1074/jbc.275.8.5560Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). In that work it was described that ZIP2-overexpressing K562 cells (human immortalized myelogenous leukemia cell line) accumulate Zn2+ in a time-, temperature-, and concentration-dependent manner, that the transport process was independent of ATP hydrolysis as well as Na+ and K+ gradients, and was stimulated in the presence of extracellular HCO3− but inhibited by lowering the extracellular pH. Altogether, these findings indicated that ZIP2 behaved as a Zn2+/HCO3− symporter (8Gaither L.A. Eide D.J. Functional expression of the human hZIP2 zinc transporter.J. Biol. Chem. 2000; 275 (10681536): 5560-556410.1074/jbc.275.8.5560Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). In contrast, prior work conducted in our laboratory suggested that ZIP2 activity is stimulated rather than inhibited by the presence of H+ (13Franz M.C. Simonin A. Graeter S. Hediger M.A. Kovacs G. Development of the first fluorescence screening assay for the SLC39A2 zinc transporter.J. Biomol. Screen. 2014; 19 (24619115): 909-91610.1177/1087057114526781Crossref PubMed Scopus (9) Google Scholar). In a more recent study, we showed that ZIP2-mediated transport in both microinjected Xenopus laevis oocytes and transiently transfected HEK293 cells is independent of both HCO3−- and H+-driving forces, but modulated by extracellular pH and voltage (14Franz M.C. Pujol-Giménez J. Montalbetti N. Fernandez-Tenorio M. DeGrado T.R. Niggli E. Romero M.F. Hediger M.A. Reassessment of the transport mechanism of the human zinc transporter SLC39A2.Biochemistry. 2018; 57 (29791142): 3976-398610.1021/acs.biochem.8b00511Crossref PubMed Scopus (17) Google Scholar). However, structural details of how H+ affects transport activity are still missing and remain to be described. Originally, ZIP2 expression was detected in uterus and prostate (8Gaither L.A. Eide D.J. Functional expression of the human hZIP2 zinc transporter.J. Biol. Chem. 2000; 275 (10681536): 5560-556410.1074/jbc.275.8.5560Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar), in the latter also at the protein level (15Desouki M.M. Geradts J. Milon B. Franklin R.B. Costello L.C. hZip2 and hZip3 zinc transporters are down regulated in human prostate adenocarcinomatous glands.Mol. Cancer. 2007; 6 (17550612): 3710.1186/1476-4598-6-37Crossref PubMed Scopus (102) Google Scholar). In prostate cancer cells, ZIP2 is down-regulated to decreased zinc levels, thereby restoring m-aconitase 2 activity and promoting ATP production via the tricarboxylic acid cycle (15Desouki M.M. Geradts J. Milon B. Franklin R.B. Costello L.C. hZip2 and hZip3 zinc transporters are down regulated in human prostate adenocarcinomatous glands.Mol. Cancer. 2007; 6 (17550612): 3710.1186/1476-4598-6-37Crossref PubMed Scopus (102) Google Scholar16Rishi I. Baidouri H. Abbasi J.A. Bullard-Dillard R. Kajdacsy-Balla A. Pestaner J.P. Skacel M. Tubbs R. Bagasra O. Prostate cancer in African American men is associated with downregulation of zinc transporters.Appl. Immunohistochem. Mol. Morphol. 2003; 11 (12966353): 253-260Crossref PubMed Scopus (69) Google Scholar, 17Franz M.C. Anderle P. Bürzle M. Suzuki Y. Freeman M.R. Hediger M.A. Kovacs G. Zinc transporters in prostate cancer.Mol. Aspects Med. 2013; 34 (23506906): 735-74110.1016/j.mam.2012.11.007Crossref PubMed Scopus (70) Google Scholar, 18Costello L.C. Franklin R.B. Zou J. Feng P. Bok R. Swanson M.G. Kurhanewicz J. Human prostate cancer ZIP1/zinc/citrate genetic/metabolic relationship in the TRAMP prostate cancer animal model.Cancer Biol. Ther. 2011; 12 (22156800): 1078-108410.4161/cbt.12.12.18367Crossref PubMed Scopus (40) Google Scholar, 19Franklin R.B. Feng P. Milon B. Desouki M.M. Singh K.K. Kajdacsy-Balla A. Bagasra O. Costello L.C. hZIP1 zinc uptake transporter down regulation and zinc depletion in prostate cancer.Mol. Cancer. 2005; 4 (16153295): 3210.1186/1476-4598-4-32Crossref PubMed Scopus (190) Google Scholar20Johnson L.A. Kanak M.A. Kajdacsy-Balla A. Pestaner J.P. Bagasra O. Differential zinc accumulation and expression of human zinc transporter 1 (hZIP1) in prostate glands.Methods. 2010; 52 (20705137): 316-32110.1016/j.ymeth.2010.08.004Crossref PubMed Scopus (46) Google Scholar). Similarly, low zinc levels and increased ZIP2 expression have also been linked to pulmonary inflammatory diseases (21Tao Y.T. Huang Q. Jiang Y.L. Wang X.L. Sun P. Tian Y. Wu H.L. Zhang M. Meng S.B. Wang Y.S. Sun Q. Zhang L.Y. Up-regulation of Slc39A2(Zip2) mRNA in peripheral blood mononuclear cells from patients with pulmonary tuberculosis.Mol. Biol. Rep. 2013; 40 (23686108): 4979-498410.1007/s11033-013-2598-zCrossref PubMed Scopus (11) Google Scholar, 22Hamon R. Homan C.C. Tran H.B. Mukaro V.R. Lester S.E. Roscioli E. Bosco M.D. Murgia C.M. Ackland M.L. Jersmann H.P. Lang C. Zalewski P.D. Hodge S.J. Zinc and zinc transporters in macrophages and their roles in efferocytosis in COPD.PLoS ONE. 2014; 9 (25350745)e11005610.1371/journal.pone.0110056Crossref PubMed Scopus (43) Google Scholar). ZIP2 also seems to play a role upon zinc depletion. Cao et al. (23Cao J. Bobo J.A. Liuzzi J.P. Cousins R.J. Effects of intracellular zinc depletion on metallothionein and ZIP2 transporter expression and apoptosis.J. Leukoc. Biol. 2001; 70 (11590192): 559-566Crossref PubMed Google Scholar) found that zinc depletion in peripheral blood mononuclear cells and the THP-1 monocytic cell line triggered up-regulation of ZIP2 with the concomitant down-regulation of zinc-binding metallothioneins. Later studies also found ZIP2 expression in macrophages, where it was the most responsive gene upon zinc depletion (24Cousins R.J. Blanchard R.K. Popp M.P. Liu L. Cao J. Moore J.B. Green C.L. A global view of the selectivity of zinc deprivation and excess on genes expressed in human THP-1 mononuclear cells.Proc. Natl. Acad. Sci. U.S.A. 2003; 100 (12756304): 6952-695710.1073/pnas.0732111100Crossref PubMed Scopus (152) Google Scholar). Recently, in healthy human skin samples, Inoue et al. (25Inoue Y. Hasegawa S. Ban S. Yamada T. Date Y. Mizutani H. Nakata S. Tanaka M. Hirashima N. ZIP2 protein, a zinc transporter, is associated with keratinocyte differentiation.J. Biol. Chem. 2014; 289 (24936057): 21451-2146210.1074/jbc.M114.560821Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) discovered that ZIP2 is up-regulated upon the induction of differentiation in cultured keratinocytes. Interestingly, ZIP2 knockdown inhibited the differentiation of keratinocytes and consequently the formation of a three-dimensional cultured epidermis. Studies with ZIP2–KO mice did not reveal any specific phenotype. However, these mice were more susceptible to abnormal embryonic development because of zinc deficiency during pregnancy (26Hara T. Takeda T.A. Takagishi T. Fukue K. Kambe T. Fukada T. Physiological roles of zinc transporters: molecular and genetic importance in zinc homeostasis.J. Physiol. Sci. 2017; 67 (28130681): 283-30110.1007/s12576-017-0521-4Crossref PubMed Scopus (225) Google Scholar). Taken together, these physiological roles warrant a more detailed investigation of the transport and ion-coupling mechanism of subfamily II ZIP transporters, of which ZIP2 is the most well-characterized member. In this work, taking advantage of the first available human ZIP4 model (5Antala S. Ovchinnikov S. Kamisetty H. Baker D. Dempski R.E. Computation and functional studies provide a model for the structure of the zinc transporter hZIP4.J. Biol. Chem. 2015; 290 (25971965): 17796-1780510.1074/jbc.M114.617613Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), we developed a homology-based model for human ZIP2 and proposed residues that might play a role in both Zn2+-coordination and H+-mediated modulation of the transport process. Site-directed mutagenesis of the proposed amino acid residues followed by detailed functional characterization of mutants validated the proposed model and provided the first structural details of the Zn2+-coordination site and H+ modulation of the transport process mediated by human ZIP2. In addition, through comparison of our model with another model built based on the bbZIP structure, we propose a transport mechanism for H+-sensitive transport of divalent metal ions and functional hot spots within the protein structure. For starting our structure–function studies, we have chosen to construct a homology-based model of human ZIP2 based on a previously published structure of human ZIP4 that was derived from the analysis of co-evolving residue pairs in the protein family (5Antala S. Ovchinnikov S. Kamisetty H. Baker D. Dempski R.E. Computation and functional studies provide a model for the structure of the zinc transporter hZIP4.J. Biol. Chem. 2015; 290 (25971965): 17796-1780510.1074/jbc.M114.617613Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The objective of our work was to identify residues within the transmembrane region that could potentially have functional roles in transport. Based on our structural model, we could identify a set of residues that possibly form the substrate-binding site (His-175, His-202, and Glu-179; Fig. 1A) based on ZIP4 data (5Antala S. Ovchinnikov S. Kamisetty H. Baker D. Dempski R.E. Computation and functional studies provide a model for the structure of the zinc transporter hZIP4.J. Biol. Chem. 2015; 290 (25971965): 17796-1780510.1074/jbc.M114.617613Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). These residues seem to be conserved in the human SLC39 family (Fig. 1B). Because Zn2+ usually binds in a tetrahedral coordination geometry, we were also interested to test whether two nearby residues, Phe-269 and Ser-176, contribute to Zn2+ binding during the transport process. A series of titratable residues was also identified on TMH 2 (His-63, Glu-67, Glu-70, and Glu-71; Fig. 1A), which we suspected might play a role in substrate recruitment to the binding site, or pH sensitivity of transport previously described by our group (14Franz M.C. Pujol-Giménez J. Montalbetti N. Fernandez-Tenorio M. DeGrado T.R. Niggli E. Romero M.F. Hediger M.A. Reassessment of the transport mechanism of the human zinc transporter SLC39A2.Biochemistry. 2018; 57 (29791142): 3976-398610.1021/acs.biochem.8b00511Crossref PubMed Scopus (17) Google Scholar). Additionally, two acidic residues (Glu-106 and Glu-120) were identified, which extend toward the membrane bilayer (Fig. 1A). Their unusual orientation prompted us to add them to the list of residues whose role in transport function we wanted to test. We next sought to determine the impact of the selected residues on transport activity and pH-dependence of human ZIP2. To this end, selected amino acid residues were substituted by either alanine or, in the case of acidic residues, the protonation-mimicking side-chain glutamine. For His-202, the glutamate substitution was also probed with the anticipation that it might alter transport selectivity, as glutamate is the corresponding residue in human ZIP8 and ZIP14 (Fig. 1B), which have been shown to transport Fe2+ in addition to Zn2+ (27Wang C.Y. Jenkitkasemwong S. Duarte S. Sparkman B.K. Shawki A. Mackenzie B. Knutson M.D. ZIP8 is an iron and zinc transporter whose cell-surface expression is up-regulated by cellular iron loading.J. Biol. Chem. 2012; 287 (22898811): 34032-3404310.1074/jbc.M112.367284Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 28Jenkitkasemwong S. Wang C.Y. Mackenzie B. Knutson M.D. Physiologic implications of metal-ion transport by ZIP14 and ZIP8.Biometals. 2012; 25 (22318508): 643-65510.1007/s10534-012-9526-xCrossref PubMed Scopus (169) Google Scholar). In the case of Phe-269, which we anticipated might interact with the divalent metal ion substrate via cation–π interactions, the leucine substitution was also tested, because it is a similarly-sized hydrophobic side chain that lacks the aromatic ring and thus is unable to form cation–π interactions. The effects of the introduced single-point mutations on the basal plasma membrane expression of human ZIP2 were assessed by immunoblotting membrane protein samples isolated by surface biotinylation. All the generated mutant variants were found to be expressed in the plasma membranes of transiently transfected HEK293 cells; however, some of them showed lower expression levels than WT ZIP2 (Fig. 2, A and E). Consequently, in all subsequent functional experiments the obtained results for each of the tested mutants were normalized to their respective average plasma membrane surface expression levels (Fig. 2, D and H). Next, the functional activities of the generated ZIP2 variants were studied using our fluorescence-based Cd2+-flux assay (Fig. 3) (13Franz M.C. Simonin A. Graeter S. Hediger M.A. Kovacs G. Development of the first fluorescence screening assay for the SLC39A2 zinc transporter.J. Biomol. Screen. 2014; 19 (24619115): 909-91610.1177/1087057114526781Crossref PubMed Scopus (9) Google Scholar, 14Franz M.C. Pujol-Giménez J. Montalbetti N. Fernandez-Tenorio M. DeGrado T.R. Niggli E. Romero M.F. Hediger M.A. Reassessment of the transport mechanism of the human zinc transporter SLC39A2.Biochemistry. 2018; 57 (29791142): 3976-398610.1021/acs.biochem.8b00511Crossref PubMed Scopus (17) Google Scholar). All the mutants generated to identify possible H+-interaction sites showed altered transport activity (Fig. 3A). Remarkably, mutants H63A and E67A and to a lesser extent E106A showed a decrease of ∼60, 35, and 30% in Cd2+ transport, respectively, whereas mutants E70A and E106Q showed an increase of ∼65 and 35% in Cd2+ transport, respectively. These findings are compatible with a role of these amino acid residues in modulating the effect of extracellular H+ on the ZIP2 transport. With respect to amino acid residues proposed as part of the substrate-binding site (Fig. 3B), our results indicate that amino acids His-175, Glu-179, and His-202 might indeed be relevant for substrate binding, but a role for Phe-269 and especially for Ser-176 seems less likely. Mutations of His-175 and His-202 to alanine were completely inactive, but the E179A variant showed a highly significant reduction of Cd2+ transport (∼60%). Interestingly, the mutation to glutamine at position 179 completely blocked the transport activity, highlighting the restrictive character of the physicochemical properties required at this position to allow substrate transport. In contrast, conservative mutation of amino acid His-202 to glutamate rescued the activity of ZIP2 by ∼60% if compared with the alanine variant. Mutation to alanine at Ser-176 did not alter transport activity, whereas in the case of F269A variant, the transport activity was reduced by ∼40%. Replacement of Phe-269 by leucine also did not alter transport activity, indicating that the presence of an aromatic residue at that position is not essential for the transport process. To verify whether the introduced mutations have a direct effect on the binding of the substrate by human ZIP2, kinetic studies of the Cd2+ transport (at extracellular pH 6.5) were conducted. ZIP2 showed an apparent affinity for Cd2+ of ∼1.46 μm (Fig. 4A, lower panel), whereas the response of the mock-transfected cells to increasing extracellular [Cd2+] lacked Michaelis-Menten kinetics (Fig. 4A, upper panel). As expected, when compared with ZIP2, the transport kinetics of the mutants generated for the residues proposed as part of the H+-interacting sites (Fig. 4B) showed differences in the Vmax values but not in the affinity for Cd2+, showing Km values in a similar range to that of WT ZIP2 (Fig. 4C). Concerning the mutants of positions predicted as part of the substrate-binding site (Fig. 4D), mutants H175A, E179Q, and H202A were not active within the range of tested extracellular [Cd2+], supporting the relevance of this cluster of residues for the substrate binding. In contrast, mutants H202E, F269A, and F269L had transport kinetics very similar to those observed for WT ZIP2 (Fig. 4E). Interestingly, while still active, the E179A variant displayed a lower apparent affinity for Cd2+ of ∼6.78 μm, indicating that the titratable side chain of Glu-179 i" @default.
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• W2923141331 date "2019-05-01" @default.
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