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• W1509744189 abstract "Members of the Zrt and Irt protein (ZIP) family are a central participant in transition metal homeostasis as they function to increase the cytosolic concentration of zinc and/or iron. However, the lack of a crystal structure hinders elucidation of the molecular mechanism of ZIP proteins. Here, we employed GREMLIN, a co-evolution-based contact prediction approach in conjunction with the Rosetta structure prediction program to construct a structural model of the human (h) ZIP4 transporter. The predicted contact data are best fit by modeling hZIP4 as a dimer. Mutagenesis of residues that comprise a central putative hZIP4 transmembrane transition metal coordination site in the structural model alter the kinetics and specificity of hZIP4. Comparison of the hZIP4 dimer model to all known membrane protein structures identifies the 12-transmembrane monomeric Piriformospora indica phosphate transporter (PiPT), a member of the major facilitator superfamily (MFS), as a likely structural homolog. Members of the Zrt and Irt protein (ZIP) family are a central participant in transition metal homeostasis as they function to increase the cytosolic concentration of zinc and/or iron. However, the lack of a crystal structure hinders elucidation of the molecular mechanism of ZIP proteins. Here, we employed GREMLIN, a co-evolution-based contact prediction approach in conjunction with the Rosetta structure prediction program to construct a structural model of the human (h) ZIP4 transporter. The predicted contact data are best fit by modeling hZIP4 as a dimer. Mutagenesis of residues that comprise a central putative hZIP4 transmembrane transition metal coordination site in the structural model alter the kinetics and specificity of hZIP4. Comparison of the hZIP4 dimer model to all known membrane protein structures identifies the 12-transmembrane monomeric Piriformospora indica phosphate transporter (PiPT), a member of the major facilitator superfamily (MFS), as a likely structural homolog. The zinc- and iron-regulated transport protein (ZIP) 3The abbreviations used are: ZIPZrt and Irt protein familyhZIP4human ZIP4TMtransmembrane domainCDFcation diffusion facilitatorSLCsolute carrierMFSmajor facilitator superfamilyORIoocyte Ringer solution. family functions to increase the cytosolic concentration of first row transition metals (1Eide D.J. The SLC39 family of metal ion transporters.Pflugers Arch. 2004; 447: 796-800Crossref PubMed Scopus (303) Google Scholar, 2Dempski R.E. The cation selectivity of the ZIP transporters.Curr. Top. Membr. 2012; 69: 221-245Crossref PubMed Scopus (35) Google Scholar). Although mutations to these proteins result in cellular zinc and/or iron deficiency, analysis of in situ experiments have demonstrated that ZIP transporters can also transport Ni2+, Cu2+, and/or Cd2+ (3Antala S. Dempski R.E. The human ZIP4 transporter has two distinct binding affinities and mediates transport of multiple transition metals.Biochemistry. 2012; 51: 963-973Crossref PubMed Scopus (56) Google Scholar, 4Pinilla-Tenas J.J. Sparkman B.K. Shawki A. Illing A.C. Mitchell C.J. Zhao N. Liuzzi J.P. Cousins R.J. Knutson M.D. Mackenzie B. Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron.Am. J. Physiol. Cell Physiol. 2011; 301: C862-C871Crossref PubMed Scopus (156) Google Scholar). ZIP member proteins have eight transmembrane domains and can be further classified into subfamilies (ZIPI, ZIPII, gufA, and LIV-1) based on sequence alignments (5Gaither L.A. Eide D.J. Eukaryotic zinc transporters and their regulation.Biometals. 2001; 14: 251-270Crossref PubMed Scopus (426) Google Scholar). The largest subfamily, LIV-1, is distinct from other ZIP proteins as its members encode a highly conserved sequence (HSVFEGLAVGIQ) in transmembrane domain (TM) 4 that has been proposed to be important for transition metal transport (6Dufner-Beattie J. Langmade S.J. Wang F. Eide D. Andrews G.K. Structure, function, and regulation of a subfamily of mouse zinc transporter genes.J. Biol. Chem. 2003; 278: 50142-50150Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The plasma membrane human (h) ZIP4 protein was the first human member of the LIV-1 subfamily of proteins to be identified as mutations within this protein lead to the zinc deficiency disease acrodermatitis enteropathica (6Dufner-Beattie J. Langmade S.J. Wang F. Eide D. Andrews G.K. Structure, function, and regulation of a subfamily of mouse zinc transporter genes.J. Biol. Chem. 2003; 278: 50142-50150Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 7Wang 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 (399) Google Scholar, 8Kury S. Dreno B. Bezieau S. Giraudet S. Kharfi M. Kamoun R. Moison J.P. Identification of SLC39A4, a gene involved in acrodermatitis enteropathica.Nat. Genet. 2002; 31: 239-240Crossref PubMed Scopus (422) Google Scholar, 9Dufner-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 (232) Google Scholar). Zrt and Irt protein family human ZIP4 transmembrane domain cation diffusion facilitator solute carrier major facilitator superfamily oocyte Ringer solution. The Znt family of proteins functions in opposition to ZIP member proteins as they act to decrease the cytosolic concentration of zinc (10Kambe T. Molecular architecture and function of ZnT transporters.in: Lutsenko S. Arguello J.M. Metal Transporters. Academic Press, San Diego, CA2012: 199-220Crossref Scopus (65) Google Scholar). Elucidation of the crystal structure of the bacterial Znt protein, YiiP, and subsequent functional studies have provided insight into the molecular details of transition metal transport mediated by Znt proteins (11Coudray N. Valvo S. Hu M. Lasala R. Kim C. Vink M. Zhou M. Provasi D. Filizola M. Tao J. Fang J. Penczek P.A. Ubarretxena-Belandia I. Stokes D.L. Inward-facing conformation of the zinc transporter YiiP revealed by cryoelectron microscopy.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 2140-2145Crossref PubMed Scopus (73) Google Scholar, 12Hoch E. Lin W. Chai J. Hershfinkel M. Fu D. Sekler I. Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 7202-7207Crossref PubMed Scopus (97) Google Scholar13Lu M. Fu D. Structure of the zinc transporter YiiP.Science. 2007; 317: 1746-1748Crossref PubMed Scopus (298) Google Scholar). The six TM YiiP protein encodes a TM transition metal coordination site and is dimeric, and both termini are cytoplasmic. Znt transporters belong to the cation diffusion facilitator (CDF) family. CDF proteins are found in bacteria, archaea, and eukaryotes (14Paulsen I.T. Saier Jr., M.H. A novel family of ubiquitous heavy metal ion transport proteins.J. Membr. Biol. 1997; 156: 99-103Crossref PubMed Scopus (295) Google Scholar). Member transporters translocate first and/or second row transition metals (2Dempski R.E. The cation selectivity of the ZIP transporters.Curr. Top. Membr. 2012; 69: 221-245Crossref PubMed Scopus (35) Google Scholar, 10Kambe T. Molecular architecture and function of ZnT transporters.in: Lutsenko S. Arguello J.M. Metal Transporters. Academic Press, San Diego, CA2012: 199-220Crossref Scopus (65) Google Scholar). Both Znt and ZIP proteins are part of a larger group of transport proteins, named solute carrier (SLC) proteins. This group of proteins includes 52 families that function as facilitative or secondary active transporters, such as the major facilitator superfamily (MFS) (15Hediger M.A. Romero M.F. Peng J.B. Rolfs A. Takanaga H. Bruford E.A. The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteinsIntroduction.Pflugers Arch. 2004; 447: 465-468Crossref PubMed Scopus (740) Google Scholar, 16Pao S.S. Paulsen I.T. Saier M.H. Major facilitator superfamily.Microbiol. Mol. Biol. Rev. 1998; 62: 1-34Crossref PubMed Google Scholar). MFS transporters encode 12 TMs and transport small solutes in response to chemiosmotic gradients. Although mechanistic linkages among family members are being studied, structural linkages between families of SLC group proteins are sparse. The absence of structural information for ZIP transporters has limited the scope and impact of mechanistic studies as it is not possible to decipher whether mutations that affect the kinetics and/or cation selectivity of ZIP proteins directly or indirectly affect transport function. Thus, the molecular mechanism of transition metal transport mediated by ZIP proteins is not resolved. In this study, we take advantage of recent progress in predicting residue pair contacts in a protein structure using co-evolution data to generate the first molecular model of hZIP4 using Rosetta in combination with co-evolution data (17Simons K.T. Ruczinski I. Kooperberg C. Fox B.A. Bystroff C. Baker D. Improved recognition of native-like protein structures using a combination of sequence-dependent and sequence-independent features of proteins.Proteins. 1999; 34: 82-95Crossref PubMed Scopus (374) Google Scholar, 18Kamisetty H. Ovchinnikov S. Baker D. Assessing the utility of coevolution-based residue-residue contact predictions in a sequence- and structure-rich era.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 15674-15679Crossref PubMed Scopus (442) Google Scholar). Mutagenesis studies that probe residues important for transition metal translocation and specificity are consistent with the model of hZIP4. Furthermore, the model has a similarity to the central transition metal coordination site of the Znt exporter, YiiP (11Coudray N. Valvo S. Hu M. Lasala R. Kim C. Vink M. Zhou M. Provasi D. Filizola M. Tao J. Fang J. Penczek P.A. Ubarretxena-Belandia I. Stokes D.L. Inward-facing conformation of the zinc transporter YiiP revealed by cryoelectron microscopy.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 2140-2145Crossref PubMed Scopus (73) Google Scholar, 13Lu M. Fu D. Structure of the zinc transporter YiiP.Science. 2007; 317: 1746-1748Crossref PubMed Scopus (298) Google Scholar). Comparison of these structures suggests that the Znt and ZIP families may share a common metal selectivity for zinc. The SP6 mMESSAGE mMACHINE kit was acquired from Invitrogen. Restriction enzymes were purchased from New England Biolabs (Ipswich, MA). The XL-1 Blue supercompetent cells were obtained from Agilent (Santa Clara, CA). Pfu polymerase was procured from Stratagene (La Jolla, CA). The radioisotopes 65ZnCl2 and 59FeCl3 were purchased from PerkinElmer. All transition metal chloride salts were purchased from Alfa Aesar (Ward Hill, MA). Additional chemicals were purchased from Sigma-Aldrich unless otherwise indicated. GREMLIN, a contact prediction method that utilizes co-evolution, was used to predict residue-residue contacts in hZIP4. GREMLIN constructs a global statistical model that simultaneously captures the conservation and co-evolution patterns in the input alignment. Strongly co-evolving residue pairs as identified by this approach are highly likely to be in contact with each other in the three-dimensional structure (18Kamisetty H. Ovchinnikov S. Baker D. Assessing the utility of coevolution-based residue-residue contact predictions in a sequence- and structure-rich era.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 15674-15679Crossref PubMed Scopus (442) Google Scholar). The input sequence of hZIP4 was trimmed to only include the following residues: 328–427 and 487–642. This trimmed sequence excludes the extracellular N-terminal domain and the only significant intracellular domain between TM3 and TM4. Both of these domains have substantial variation between species. A multiple sequence alignment was generated using HHblits (19Söding J. Protein homology detection by HMM-HMM comparison.Bioinformatics. 2005; 21: 951-960Crossref PubMed Scopus (1859) Google Scholar) with the following options: n, 8; e, 1 × 10−20; maxfilt, ∞; neffmax, 20; nodiff, realign_max ∞. The resulting alignment was then filtered to exclude any sequence that did not cover at least 50% of the query and to reduce the sequence redundancy to 90%. GREMLIN was then run with default parameters. The standard Rosetta ab initio structure prediction method was used to model the three-dimensional structure of hZIP4 trimmed to only include residues 328–648. The default Rosetta energy function was modified to enable membrane specific terms with the following weights: fa_sol, 0.0; fa_mbenv, 0.3; fa_mbsolv, 0.35; and Menv_smooth, 0.5 (20Barth P. Schonbrun J. Baker D. Toward high-resolution prediction and design of transmembrane helical protein structures.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 15682-15687Crossref PubMed Scopus (182) Google Scholar, 21Yarov-Yarovoy V. DeCaen P.G. Westenbroek R.E. Pan C.Y. Scheuer T. Baker D. Catterall W.A. Structural basis for gating charge movement in the voltage sensor of a sodium channel.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: E93-102Crossref PubMed Scopus (179) Google Scholar). Transmembrane spans (residues 2–23, 33–53, 74–95, 167–191, 197–222, 228–252, 259–282, and 290–314) were defined using the consensus output of the MESSA server (22Cong Q. Grishin N.V. MESSA: MEta-Server for protein Sequence Analysis.BMC Biol. 2012; 10: 82Crossref PubMed Scopus (36) Google Scholar). To reduce the sampling space, sigmoidal restraints that modified the energy function were introduced. The shape of the sigmoid is defined using a distance cutoff, the slope, the intercept, and the strength of the weight parameter (Fig. 1A). The Rosetta ab initio protocol consists of two stages. In the initial stage (“centroid”), side chains are represented by fixed center-of-mass atoms allowing for rapid generation and evaluation of various protein-like topologies; the second stage (“full-atom”) places all-atom side chains into a starting topology and iteratively refines the model until a low energy structure is found (17Simons K.T. Ruczinski I. Kooperberg C. Fox B.A. Bystroff C. Baker D. Improved recognition of native-like protein structures using a combination of sequence-dependent and sequence-independent features of proteins.Proteins. 1999; 34: 82-95Crossref PubMed Scopus (374) Google Scholar, 23Raman S. Vernon R. Thompson J. Tyka M. Sadreyev R. Pei J. Kim D. Kellogg E. DiMaio F. Lange O. Kinch L. Sheffler W. Kim B.H. Das R. Grishin N.V. Baker D. Structure prediction for CASP8 with all-atom refinement using Rosetta.Proteins. 2009; 77: 89-9910.1002/prot.22540Crossref PubMed Scopus (348) Google Scholar). To favor sampling of topologies consistent with GREMLIN predictions, sigmoidal distance restraints (Fig. 1A) were introduced between residue pairs predicted to be in contact by GREMLIN (Fig. 2). When used in the centroid stage of Rosetta, these restraints were introduced between carbon-β atoms (carbon-α in the case of glycine), at amino acid pair-specific Cβ-Cβ cutoff and slopes, as described in Supporting Information Table 3 within Ref. 18Kamisetty H. Ovchinnikov S. Baker D. Assessing the utility of coevolution-based residue-residue contact predictions in a sequence- and structure-rich era.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 15674-15679Crossref PubMed Scopus (442) Google Scholar; in the full-atom stage, these were replaced with ambiguous distance restraints between side-chain heavy atoms (cutoff of 5.5 and slope of 4) (24Lange O.F. Rossi P. Sgourakis N.G. Song Y. Lee H.W. Aramini J.M. Ertekin A. Xiao R. Acton T.B. Montelione G.T. Baker D. Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 10873-10878Crossref PubMed Scopus (153) Google Scholar). The relative weight of each restraint was based on the GREMLIN score. The total atom pair restraint score was scaled to be roughly one-half the total Rosetta score. In addition to these restraints, additional strong repulsive distance restraints (weight 100, cutoff 35, slope 2, and intercept of 100) were added between extracellular regions (defined by residues 329, 391,522, 582, and 648) and intracellular regions (defined by residues 353, 425, 490, 553, and 615), and strong attractive restraints (weight 100, cutoff 35, slope 2 and intercept of 0) were added within intracellular regions and extracellular regions, effectively constructing a membrane-like sampling space (Fig. 1B). These restraints were introduced between pairs of Cα atoms. The top 1010 models ranked by distance restraints score, with z-score ≥2 (Fig. 3), were clustered based on structural similarity as calculated by TM-score (25Zhang Y. Skolnick J. Scoring function for automated assessment of protein structure template quality.Proteins. 2004; 57: 702-710Crossref PubMed Scopus (1236) Google Scholar) after excluding the disordered regions (positions 428–486 and 643–648) (26Bafaro E. Antala S. Nguyen T.V. Dzul S.P. Doyon B. Stemmler T.L. Dempski R.E. The large intracellular loop of hZIP4 is an intrinsically disordered zinc binding domain.Metallomics. 2015; 10.1039/C5MT00066ACrossref PubMed Google Scholar). Clusters were defined as connected components of a network, where each edge is between models with TM-score ≥0.7. The selected model was then further energy-minimized with Rosetta to remove clashes, while respecting structural symmetry and GREMLIN restraints (27DiMaio F. Leaver-Fay A. Bradley P. Baker D. André I. Modeling symmetric macromolecular structures in Rosetta3.PLoS One. 2011; 6e20450Crossref PubMed Scopus (148) Google Scholar). Preparation of plasmid construct and mRNA was performed as described previously (3Antala S. Dempski R.E. The human ZIP4 transporter has two distinct binding affinities and mediates transport of multiple transition metals.Biochemistry. 2012; 51: 963-973Crossref PubMed Scopus (56) Google Scholar). Site-directed mutagenesis was performed using QuikChange according to the manufacturer's instructions. Following mutagenesis, the entire gene was sequenced. Oocytes were extracted from X. laevis in accordance with Worcester Polytechnic Institute Animal Care and Use committee-approved protocol (3Antala S. Dempski R.E. The human ZIP4 transporter has two distinct binding affinities and mediates transport of multiple transition metals.Biochemistry. 2012; 51: 963-973Crossref PubMed Scopus (56) Google Scholar, 28Richards R. Dempski R.E. Examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling.J. Vis. Exp. 2011; 10.3791/2627Crossref PubMed Google Scholar). Oocytes were digested with collagenase (3 mg/ml) for 3 h. Following enzymatic digestion, oocytes were rinsed with ORI−Ca2+ buffer (90 mm NaCl, 2 mm KCl, and 5 mm MOPS at pH 7.4) three times and stored in ORI+Ca2+ (ORI−Ca2+ buffer with 2 mm CaCl2 and 20 μg/ml gentamycin) buffer overnight at 16 °C. mRNA or diethyl pyrocarbonate-treated water was injected into each oocyte using a micro-injector and stored in ORI+Ca2+ buffer for 3 days at 16 °C. Radioisotope uptake assays were performed 72 h following injection (3Antala S. Dempski R.E. The human ZIP4 transporter has two distinct binding affinities and mediates transport of multiple transition metals.Biochemistry. 2012; 51: 963-973Crossref PubMed Scopus (56) Google Scholar). Briefly, oocytes were washed with uptake buffer (90 mm NaCl, 10 mm HEPES, pH 7.4, and 1 mm ascorbic acid). Oocytes were incubated in uptake buffer containing 65ZnCl2 for 1 h. At the conclusion of the transport assay, oocytes were washed three times with uptake buffer and solubilized in 200 μl of 1% w/v SDS solution. Solubilized oocytes were mixed with scintillation fluid (Scintisafe-30%, Fisher Scientific) and subjected to radioactivity measurement using a Beckman LS6500 multi-purpose scintillation counter. For iron uptake assays, 59FeCl2 was added as 59FeCl3 in the presence of 1 mm ascorbic acid. For competition experiments, 3 μm 65ZnCl2 was added to 600 μm of various transition metals. For control, no additional transition metal was added. In these experiments, it is necessary to subtract diethyl pyrocarbonate/water-injected controls at each concentration of zinc during the uptake assay to eliminate background radioisotope uptake. The resulting data points were fit with the equation: y = (Vmax × [Zn2+]n)/Kmn + [Zn2+]n where Vmax is the maximal velocity, [Zn2+] is the concentration of divalent metal ion, Km is the concentration of divalent metal at one-half Vmax, and n is the Hill co-efficient. hZIP4 surface expression was detected using EZ-Link Sulfo-NHS-SS-Biotin and biotinylation kit (Thermo Scientific) according to the manufacturer's instructions. In brief, 35 oocytes 72 h after injection were washed three times with ice-cold PBS (0.1 m sodium phosphate, 0.15 m NaCl, pH 7.2) before incubating them with 2 ml of biotinylation buffer. Sulfo-NHS-SS-Biotin was dissolved in PBS to a final concentration of 0.11 mg/ml, and oocytes were incubated with biotinylation buffer for 90 min at 4 °C with gentle shaking. The reaction was stopped upon the addition of 200 μl of quenching solution. Biotin-labeled oocytes were washed four times with TBS (0.025 m Tris, 0.15 m NaCl, pH 7.2). Finally, oocytes were resuspended in 500 μl of solubilization buffer (TBS buffer containing 1% (w/v) n-dodecyl-β-d-maltoside and 1 mm PMSF). Oocytes were homogenized by passing them through a 25-gauge needle. The lysate was centrifuged at 14,000 × g for 20 min at 4 °C. The supernatant was collected into microcentrifuge tubes. The pellet was resuspended in solubilization buffer and centrifuged at 14,000 × g for 20 min at 4 °C. The supernatants were mixed and incubated with 50 μl of NeutrAvidin resin at room temperature for 60 min with gentle shaking. The resin was washed four times with solubilization buffer. Finally, biotinylated proteins were eluted by boiling resin with Laemmli buffer for 20 min. The eluted biotinylated proteins were separated on SDS-PAGE. hZIP4 was detected by Western blot with a rabbit polyclonal antibody raised against the N terminus of hZIP4 (Aviva Systems Biology, San Diego, CA). Loosely adapting the protocol from Dürr et al. (29Dürr K.L. Tavraz N.N. Dempski R.E. Bamberg E. Friedrich T. Functional significance of E2 state stabilization by specific α/β-subunit interactions of Na,K- and H,K-ATPase.J. Biol. Chem. 2009; 284: 3842-3854Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), 50 oocytes were added to breaking buffer (20 mm HEPES, pH 7.4, 150 mm NaCl and 1 mm PMSF) and passed through a 25-gauge needle followed by 1 min of low speed centrifugation (100 × g and 4 °C). The supernatant was collected in a separate microcentrifuge tube and centrifuged (1 min at 100 × g and 4 °C). The procedure was repeated until no pellet was observed upon centrifugation. The supernatant was then spun down at 14,000 × g for 30 min at 4 °C. The membrane pellet was resuspended in solubilizing buffer (20 mm HEPES, pH 7.4, 150 mm NaCl, 1% w/v n-dodecyl-β-d-maltoside, and 1 mm PMSF) and incubated at room temperature for 5 min. The insoluble fraction was separated by high speed centrifugation (14,000 × g for 30 min at 4 °C). The supernatant was added into Laemmli buffer in the absence of reducing agent. The solubilized total membrane proteins were separated on SDS-PAGE. The Western blot was developed using rabbit polyclonal antibody raised against N terminus of hZIP4 (Aviva Systems Biology). A model of hZIP4 was generated using predicted contacts and Rosetta (18Kamisetty H. Ovchinnikov S. Baker D. Assessing the utility of coevolution-based residue-residue contact predictions in a sequence- and structure-rich era.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 15674-15679Crossref PubMed Scopus (442) Google Scholar). Functional studies support this model. The structure comparison method was then used to compare the hZIP4 structural model with non-redundant Protein Data Bank (PDB) membrane protein structures (30Xu J. Zhang Y. How significant is a protein structure similarity with TM-score = 0.5?.Bioinformatics. 2010; 26: 889-895Crossref PubMed Scopus (481) Google Scholar, 31Zhang Y. Skolnick J. TM-align: a protein structure alignment algorithm based on the TM-score.Nucleic Acids Res. 2005; 33: 2302-2309Crossref PubMed Scopus (1907) Google Scholar). Membrane structures have been modeled using contact prediction based on co-evolutionary patterns in large multiple sequence alignments of homologous proteins (32Hopf T.A. Colwell L.J. Sheridan R. Rost B. Sander C. Marks D.S. Three-dimensional structures of membrane proteins from genomic sequencing.Cell. 2012; 149: 1607-1621Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 33Nugent T. Jones D.T. Accurate de novo structure prediction of large transmembrane protein domains using fragment-assembly and correlated mutation analysis.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: E1540-E1547Crossref PubMed Scopus (155) Google Scholar). To see whether such an analysis was possible for hZIP4, we constructed an alignment over the conserved transmembrane region by querying the UniProt database with HHblits (19Söding J. Protein homology detection by HMM-HMM comparison.Bioinformatics. 2005; 21: 951-960Crossref PubMed Scopus (1859) Google Scholar, 34Wu C.H. Apweiler R. Bairoch A. Natale D.A. Barker W.C. Boeckmann B. Ferro S. Gasteiger E. Huang H. Lopez R. Magrane M. Martin M.J. Mazumder R. O'Donovan C. Redaschi N. Suzek B. The Universal Protein Resource (UniProt): an expanding universe of protein information.Nucleic Acids Res. 2006; 34: D187-D191Crossref PubMed Scopus (882) Google Scholar). The resulting non-redundant alignment contained 1731 homologous sequences, 6.8 sequences per length of 255, which is more than the minimum sequences (5 sequences per length) required to predict residue pairs in contact in the three-dimensional structure of hZIP4 using GREMLIN, a co-evolution-based contact prediction approach that is more accurate than other extant approaches (18Kamisetty H. Ovchinnikov S. Baker D. Assessing the utility of coevolution-based residue-residue contact predictions in a sequence- and structure-rich era.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 15674-15679Crossref PubMed Scopus (442) Google Scholar). These predictions were then used as distance restraints in the Rosetta ab initio protocol along with other membrane-specific terms to generate over 100,000 structural models of hZIP4 using a distributed computing network, [email protected] Of these, the top 1010 models based on restraint score were extracted. These models formed three large clusters (Fig. 4), of which one cluster agreed with GREMLIN predictions and formed physically realistic structures. Despite this extensive sampling, we observed one set of predicted contacts that were consistently not made by the structural models (Fig. 4A). Further inspection revealed that it was physically impossible to make all these contacts in regions between TM2–3 and TM7–8, without breaking contacts between other helices in a monomeric state. The top 384 (1.5 × length of query) co-evolving pairs of residues that were at least three residues apart (Fig. 2) in the protein sequence were then selected to be used in subsequent structural modeling in Rosetta. Using these top co-evolving pairs, an oligomeric interface could be readily made (Figs. 5, B and C, and 6). Notable for these results is that each of the variants in cluster within Fig. 4A can make the dimer interface as shown in Fig. 6. Equally, these models have histidine residues lining the transmembrane core forming a potential permeating pathway (Fig. 5A).FIGURE 6Variations within cluster A are consistent within context of a dimer. When we examine variation within cluster A, we find that the models are capable of making the remainder of the top co-evolving residues in the context of a dimer. For example, if we reconnect the loop regions between helices 2 and 3, swapping helix 3 between the homo-dimer (A ≥ B), this preserves all predicted helical contacts. Another example would be to reconnect the loop between helices 7 and 8, swapping helix 8 (A ≥ C), or a combination of both (A ≥ D). These swaps are represented in the variation of the top cluster (Fig. 4A).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our laboratory has demonstrated that covalent labeling of histidine, but not cysteine residues, decreases the velocity of Zn2+ transport (3Antala S. Dempski R.E. The human ZIP4 transporter has two distinct binding affinities and mediates transport of multiple transition metals.Biochemistry. 2012; 51: 963-973Crossref PubMed Scopus (56) Google Scholar). Independent of the modeling studies, we examined the functional role of each transmembrane histidine residue in transition metal translocation. hZIP4 encodes six transmembrane histidine residues: 379 (TM2), 507 (TM4), 536, 540, and 550 (TM5), as well as 624 (TM8). We individually replaced each TM histidine residue with alanine. Zinc transport kinetics were determined for each of the mutant transporters using our established uptake assay (3Antala S. Dempski R.E. The human ZIP4 transporter has two distinct binding affinities and mediates transport of multiple transition metals.Biochemistry. 2012; 51: 963-973Crossref PubMed Scopus (56) Google Scholar). Previously, we have demonstrated that the wild type hZIP4 has t" @default.
• W1509744189 created "2016-06-24" @default.
• W1509744189 creator A5053192926 @default.
• W1509744189 creator A5058769019 @default.
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• W1509744189 date "2015-07-01" @default.
• W1509744189 modified "2023-10-05" @default.
• W1509744189 title "Computation and Functional Studies Provide a Model for the Structure of the Zinc Transporter hZIP4" @default.
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