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Evolutionary analysis of Slc11 mechanism of proton-coupled metal-ion transmembrane import

Inrs-Institut Armand-Frappier, 531, Bd des prairies, Laval, QC H7V 1B7, Canada

Topical Section: Membrane protein structural biology

Determination of the crystal structure of ScaDMT, a member of the Slc11 family, provided opportunity to advance understanding of proton-dependent metal-ion uptake by interfacing Slc11 molecular evolution and structural biology. Slc11 carriers belong to the ancient and broadly distributed APC superfamily characterized by the pseudo-symmetric LeuT-fold. This fold comprises two topologically inverted repeats (protomers) that exchange alternate configurations during carrier cycling. Examining ScaDMT molecule inserted within a model membrane allowed to pinpoint residues that may interact with surrounding lipid solvent molecules. Three-dimensional mapping of Slc11-specific sites demonstrated they distribute at the protomer interface, along the transmembrane ion-conduction pathway. Functional sites were predicted by modeling hypothetical ScaDMT alternate conformers based on APC templates; these candidate homologous sites were found to co-localize with Slc11-specific sites, a distribution pattern that fits the functional diversity in the APC superfamily. Sites that diverged among eukaryotic Slc11 (Nramp) types were located in transmembrane helices that may participate in discrete steps during co-substrate translocation, suggesting these sites influence transport activity. Adding some functional dimension to Slc11 carrier evolution will inform molecular understanding of metal-ion transport selectivity and regulation, Slc11 physiological roles and contribution to host resistance to microbial infection.
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Keywords metal-ion; membrane transport; proton-coupling; solute carrier family 11; proton-dependent Mn transporter; natural resistance-associated macrophage protein; divalent metal transporter; LeuT fold; amino acid-polyamine-organo-cation superfamily.

Citation: Mathieu F. M. Cellier. Evolutionary analysis of Slc11 mechanism of proton-coupled metal-ion transmembrane import. AIMS Biophysics, 2016, 3(2): 286-318. doi: 10.3934/biophy.2016.2.286


  • 1. Hediger MA, Clemencon B, Burrier RE, et al. (2013) The ABCs of membrane transporters in health and disease (SLC series): introduction. Mol Aspects Med 34: 95–107.    
  • 2. Illing AC, Shawki A, Cunningham CL, et al. (2012) Substrate profile and metal-ion selectivity of human divalent metal-ion transporter-1. J Biol Chem 287: 30485–30496.    
  • 3. Cellier MF (2013) Cell-Type Specific Determinants of NRAMP1 Expression in Professional Phagocytes. Biology (Basel) 2: 233–283.
  • 4. Braasch I, Gehrke AR, Smith JJ, et al. (2016) The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons. Nat Genet 48: 427–437.    
  • 5. Cellier MF, Courville P, Campion C (2007) Nramp1 phagocyte intracellular metal withdrawal defense. Microbes Infect 9: 1662–1670.    
  • 6. Vidal SM, Malo D, Vogan K, et al. (1993) Natural resistance to infection with intracellular parasites: isolation of a candidate for Bcg. Cell 73: 469–485.    
  • 7. Peracino B, Wagner C, Balest A, et al. (2006) Function and mechanism of action of Dictyostelium Nramp1 (Slc11a1) in bacterial infection. Traffic 7: 22–38.    
  • 8. Ehrnstorfer IA, Geertsma ER, Pardon E, et al. (2014) Crystal structure of a SLC11 (NRAMP) transporter reveals the basis for transition-metal ion transport. Nat Struct Mol Biol 21: 990–996.    
  • 9. Vastermark A, Wollwage S, Houle ME, et al. (2014) Expansion of the APC superfamily of secondary carriers. Proteins 82: 2797–2811.    
  • 10. Yamashita A, Singh SK, Kawate T, et al. (2005) Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters. Nature 437: 215–223.    
  • 11. Forrest LR (2015) Structural Symmetry in Membrane Proteins. Annu Rev Biophys 44: 311–337.    
  • 12. Cellier MF (2012) Nutritional immunity: homology modeling of Nramp metal import. Adv Exp Med Biol 946: 335–351.    
  • 13. Shi Y (2013) Common Folds and Transport Mechanisms of Secondary Active Transporters. Annu Rev Biophys 42: 51–72.    
  • 14. Kowalczyk L, Ratera M, Paladino A, et al. (2011) Molecular basis of substrate-induced permeation by an amino acid antiporter. Proc Natl Acad Sci USA 108: 3935–3940.    
  • 15. Krishnamurthy H, Piscitelli CL, Gouaux E (2009) Unlocking the molecular secrets of sodium-coupled transporters. Nature 459: 347–355.    
  • 16. Forrest LR, Kramer R, Ziegler C (2011) The structural basis of secondary active transport mechanisms. Biochim Biophys Acta 1807: 167–188.    
  • 17. Faham S, Watanabe A, Besserer GM, et al. (2008) The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport. Science 321: 810–814.    
  • 18. Weyand S, Shimamura T, Yajima S, et al. (2008) Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter. Science 322: 709–713.    
  • 19. Ressl S, Terwisscha van Scheltinga AC, Vonrhein C, et al. (2009) Molecular basis of transport and regulation in the Na(+)/betaine symporter BetP. Nature 458: 47–52.    
  • 20. Gao X, Lu F, Zhou L, et al. (2009) Structure and mechanism of an amino acid antiporter. Science 324: 1565–1568.    
  • 21. Shaffer PL, Goehring A, Shankaranarayanan A, et al. (2009) Structure and mechanism of a na+-independent amino Acid transporter. Science 325: 1010–1014.    
  • 22. Fang Y, Jayaram H, Shane T, et al. (2009) Structure of a prokaryotic virtual proton pump at 3.2 A resolution. Nature 460: 1040–1043.
  • 23. Gao X, Zhou L, Jiao X, et al. (2010) Mechanism of substrate recognition and transport by an amino acid antiporter. Nature 463: 828–832.    
  • 24. Tang L, Bai L, Wang WH, et al. (2010) Crystal structure of the carnitine transporter and insights into the antiport mechanism. Nat Struct Mol Biol 17: 492–496.    
  • 25. Shimamura T, Weyand S, Beckstein O, et al. (2010) Molecular basis of alternating access membrane transport by the sodium-hydantoin transporter Mhp1. Science 328: 470–473.    
  • 26. Schulze S, Koster S, Geldmacher U, et al. (2010) Structural basis of Na(+)-independent and cooperative substrate/product antiport in CaiT. Nature 467: 233–236.    
  • 27. Krishnamurthy H, Gouaux E (2012) X-ray structures of LeuT in substrate-free outward-open and apo inward-open states. Nature 481: 469–474.    
  • 28. Ma D, Lu P, Yan C, et al. (2012) Structure and mechanism of a glutamate-GABA antiporter. Nature 483: 632–636.    
  • 29. Khafizov K, Perez C, Koshy C, et al. (2012) Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP. Proc Natl Acad Sci USA 109: E3035–E3044.    
  • 30. Perez C, Faust B, Mehdipour AR, et al. (2014) Substrate-bound outward-open state of the betaine transporter BetP provides insights into Na+ coupling. Nat Commun 5: 4231.
  • 31. Malinauskaite L, Quick M, Reinhard L, et al. (2014) A mechanism for intracellular release of Na+ by neurotransmitter/sodium symporters. Nat Struct Mol Biol 21: 1006–1012.    
  • 32. Chaloupka R, Courville P, Veyrier F, et al. (2005) Identification of functional amino acids in the Nramp family by a combination of evolutionary analysis and biophysical studies of metal and proton cotransport in vivo. Biochemistry 44: 726–733.    
  • 33. Gaucher EA, Gu X, Miyamoto MM, et al. (2002) Predicting functional divergence in protein evolution by site-specific rate shifts. Trends Biochem Sci 27: 315–321.    
  • 34. Knudsen B, Miyamoto MM, Laipis PJ, et al. (2003) Using Evolutionary Rates to Investigate Protein Functional Divergence and Conservation. A case study of the carbonic anhydrases. Genetics 164: 1261–1269.
  • 35. Gu X, Vander Velden K (2002) DIVERGE: phylogeny-based analysis for functional-structural divergence of a protein family. Bioinformatics 18: 500–501.
  • 36. Gu X, Zou Y, Su Z, et al. (2013) An update of DIVERGE software for functional divergence analysis of protein family. Mol Biol Evol 30: 1713–1719.
  • 37. Echave J, Spielman SJ, Wilke CO (2016) Causes of evolutionary rate variation among protein sites. Nat Rev Genet 17: 109–121.    
  • 38. Zhang J, Yang JR (2015) Determinants of the rate of protein sequence evolution. Nat Rev Genet 16: 409–420.    
  • 39. Sandler I, Zigdon N, Levy E, et al. (2014) The functional importance of co-evolving residues in proteins. Cell Mol Life Sci 71: 673–682.    
  • 40. de Juan D, Pazos F, Valencia A (2013) Emerging methods in protein co-evolution. Nat Rev Genet 14: 249–261.
  • 41. Shin JH, Wakeman CA, Goodson JR, et al. (2014) Transport of magnesium by a bacterial nramp-related gene. PLoS Genet 10: e1004429.    
  • 42. Courville P, Urbankova E, Rensing C, et al. (2008) Solute carrier 11 cations symport requires distinct residues in transmembrane helices 1 and 6. J Biol Chem 283: 9651–9658.    
  • 43. Cellier MF (2012) Nramp: from sequence to structure and mechanism of divalent metal import. Curr Top Membr 69: 249–293.    
  • 44. Richer E, Courville P, Cellier M (2004) Molecular Evolutionary Analysis of the Nramp Family. Cellier M. and Gros P. (eds) Molecular biology intelligence unit. 178–194. Springer.
  • 45. Richer E, Courville P, Bergevin I, et al. (2003) Horizontal gene transfer of "prototype" Nramp in bacteria. J Mol Evol 57: 363–376.    
  • 46. Jenuth JP (2000) The NCBI. Publicly available tools and resources on the Web. Methods Mol Biol 132: 301–312.
  • 47. Biegert A, Mayer C, Remmert M, et al. (2006) The MPI Bioinformatics Toolkit for protein sequence analysis. Nucleic Acids Res 34: W335–W339.
  • 48. Tamura K, Peterson D, Peterson N, et al. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.
  • 49. Thompson JD, Gibson TJ, Higgins DG (2002) Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinform Chapter 2: Unit 2.3.: Unit.
  • 50. Gouy M, Guindon S, Gascuel O (2010) SeaView Version 4: A Multiplatform Graphical User Interface for Sequence Alignment and Phylogenetic Tree Building. Mol Biol Evol 27: 221–224.
  • 51. Burki F (2014) The Eukaryotic Tree of Life from a Global Phylogenomic Perspective. Cold Spring Harbor Perspectives in Biology 6. a016147.
  • 52. Koonin EV (2010) The origin and early evolution of eukaryotes in the light of phylogenomics. Genome Biol 11: 209.    
  • 53. Adl SM, Simpson AGB, Lane CE, et al. (2012) The Revised Classification of Eukaryotes. J Eukaryot Microbiol 59: 429–493.    
  • 54. Lin Z, Fernandez-Robledo JA, Cellier MF, et al. (2011) The natural resistance-associated macrophage protein from the protozoan parasite Perkinsus marinus mediates iron uptake. Biochemistry 50: 6340–6355.    
  • 55. Guindon S, Dufayard JF, Lefort V, et al. (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59: 307–321.
  • 56. Shih AC, Lee DT, Peng CL, et al. (2007) Phylo-mLogo: an interactive and hierarchical multiple-logo visualization tool for alignment of many sequences. BMC Bioinformatics 8: 63.    
  • 57. Stansfeld PJ, Goose JE, Caffrey M, et al. (2015) MemProtMD: Automated Insertion of Membrane Protein Structures into Explicit Lipid Membranes. Structure 23: 1350–1361.    
  • 58. DeLano WL (2002) The PyMOL Molecular Graphics System. DeLano Scientific, San carlos, california, USA. Available from: http: //www.pymol.org.
  • 59. Ye Y, Godzik A (2003) Flexible structure alignment by chaining aligned fragment pairs allowing twists. Bioinformatics 19 Suppl 2: ii246–ii255.
  • 60. Pieper U, Webb BM, Barkan DT, et al. (2011) ModBase, a database of annotated comparative protein structure models, and associated resources. Nucleic Acids Res 39: D465–D474.
  • 61. Narunsky A, Nepomnyachiy S, Ashkenazy H, et al. (2015) ConTemplate Suggests Possible Alternative Conformations for a Query Protein of Known Structure. Structure 23: 2162–2170.    
  • 62. Czachorowski M, Lam-Yuk-Tseung S, Cellier M, et al. (2009) Transmembrane Topology of the Mammalian Slc11a2 Iron Transporter. Biochemistry 48: 8422–8434.    
  • 63. Xia J, Yamaji N, Kasai T, et al. (2010) Plasma membrane-localized transporter for aluminum in rice. Proc Natl Acad Sci USA 107: 18381–18385.    
  • 64. Pottier M, Oomen R, Picco C, et al. (2015) Identification of mutations allowing Natural Resistance Associated Macrophage Proteins (NRAMP) to discriminate against cadmium. Plant J 83: 625–637.    
  • 65. Tavoulari S, Margheritis E, Nagarajan A, et al. (2016) Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog. J Biol Chem 291: 1456–1471.    
  • 66. Ashkenazy H, Erez E, Martz E, et al. (2010) ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res 38: W529–W533.
  • 67. De Falco L, Bruno M, Andolfo I, et al. (2012) Identification and characterization of the first SLC11A2 isoform 1a mutation causing a defect in splicing process and an hypomorphic allele expression of the SLC11A2 gene. Br J Haematol 159: 492–495.    
  • 68. Bardou-Jacquet E, Island ML, Jouanolle AM, et al. (2011) A novel N491S mutation in the human SLC11A2 gene impairs protein trafficking and in association with the G212V mutation leads to microcytic anemia and liver iron overload. Blood Cells Mol Dis 47: 243–248.    
  • 69. Blanco E, Kannengiesser C, Grandchamp B, et al. (2009) Not all DMT1 mutations lead to iron overload. Blood Cells Mol Dis 43: 199–201.    
  • 70. Iolascon A, De FL (2009) Mutations in the gene encoding DMT1: clinical presentation and treatment. Semin Hematol 46: 358–370.    
  • 71. Iolascon A, d'Apolito M, Servedio V, et al. (2006) Microcytic anemia and hepatic iron overload in a child with compound heterozygous mutations in DMT1 (SCL11A2) Blood 107: 349–354.
  • 72. Iolascon A, Camaschella C, Pospisilova D, et al. (2008) Natural history of recessive inheritance of DMT1 mutations. J Pediatr 152: 136–139.    
  • 73. Mims MP, Guan Y, Pospisilova D, et al. (2005) Identification of a human mutation of DMT1 in a patient with microcytic anemia and iron overload. Blood 105: 1337–1342.
  • 74. Simmons KJ, Jackson SM, Brueckner F, et al. (2014) Molecular mechanism of ligand recognition by membrane transport protein, Mhp1. EMBO J 33: 1831–1844.    
  • 75. Alva V, Soding J, Lupas AN (2015) A vocabulary of ancient peptides at the origin of folded proteins. Elife 4.
  • 76. Das S, Dawson NL, Orengo CA (2015) Diversity in protein domain superfamilies. Curr Opin Genet Dev 35: 40–49.    
  • 77. Vergara-Jaque A, Fenollar-Ferrer C, Kaufmann D, et al. (2015) Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms. Front Pharmacol 6: 183.
  • 78. Siddle KJ, Quintana-Murci L (2014) The Red Queen's long race: human adaptation to pathogen pressure. Curr Opin Genet Dev 29: 31–38.    
  • 79. Nakashige TG, Zhang B, Krebs C, et al. (2015) Human calprotectin is an iron-sequestering host-defense protein. Nat Chem Biol 11: 765–771.    
  • 80. Becker KW, Skaar EP (2014) Metal limitation and toxicity at the interface between host and pathogen. FEMS Microbiol Rev 38: 1235–1249.    
  • 81. Lisher JP, Giedroc DP (2013) Manganese acquisition and homeostasis at the host-pathogen interface. Front Cell Infect Microbiol 3: 91.
  • 82. Ganz T (2009) Iron in innate immunity: starve the invaders. Curr Opin Immunol 21: 63–67.    
  • 83. Ganz T, Nemeth E (2015) Iron homeostasis in host defence and inflammation. Nat Rev Immunol 15: 500–510.    
  • 84. Buracco S, Peracino B, Cinquetti R, et al. (2015) Dictyostelium Nramp1, which is structurally and functionally similar to mammalian DMT1 transporter, mediates phagosomal iron efflux. J Cell Sci 128: 3304–3316.    
  • 85. Gallant CJ, Malik S, Jabado N, et al. (2007) Reduced in vitro functional activity of human NRAMP1 (SLC11A1) allele that predisposes to increased risk of pediatric tuberculosis disease. Genes Immun 8: 691–698.    
  • 86. Desiro A, Salvioli A, Ngonkeu EL, et al. (2014) Detection of a novel intracellular microbiome hosted in arbuscular mycorrhizal fungi. ISME J 8: 257–270.    
  • 87. Ohshima S, Sato Y, Fujimura R, et al. (2016) Mycoavidus cysteinexigens gen. nov., sp. nov., an endohyphal bacterium isolated from a soil isolate of the fungus Mortierella elongata. Int J Syst Evol Microbiol.
  • 88. Cohen A, Nevo Y, Nelson N (2003) The first external loop of the metal ion transporter DCT1 is involved in metal ion binding and specificity. Proc Natl Acad Sci USA 100: 10694–10699.    
  • 89. Courville P, Chaloupka R, Cellier MF (2006) Recent progress in structure-function analyses of Nramp proton-dependent metal-ion transporters. Biochem Cell Biol 84: 960–978.    
  • 90. Chen X, Peng J, Cohen A, et al. (1999) Yeast SMF1 mediates H+ -coupled iron uptake with concomitant uncoupled cation currents. J Biol Chem 274: 35089–35094.    
  • 91. Agranoff D, Collins L, Kehres D, et al. (2005) The Nramp orthologue of Cryptococcus neoformans is a pH-dependent transporter of manganese, iron, cobalt and nickel. Biochem J 385: 225–232.    
  • 92. Gunshin H, Mackenzie B, Berger UV, et al. (1997) Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 388: 482–488.    
  • 93. Sacher A, Cohen A, Nelson N (2001) Properties of the mammalian and yeast metal-ion transporters DCT1 and Smf1p expressed in Xenopus laevis oocytes. J Exp Biol 204: 1053–1061.
  • 94. Bleackley MR, MacGillivray RT (2011) Transition metal homeostasis: from yeast to human disease. Biometals 24: 785–809.    
  • 95. Martin JE, Waters LS, Storz G, et al. (2015) The Escherichia coli small protein MntS and exporter MntP optimize the intracellular concentration of manganese. PLoS Genet 11: e1004977.    
  • 96. Tsai MF, Fang Y, Miller C (2012) Sided functions of an arginine-agmatine antiporter oriented in liposomes. Biochemistry 51: 1577–1585.    
  • 97. Nevo Y, Nelson N (2006) The NRAMP family of metal-ion transporters. Biochim Biophys Acta 1763: 609–620.    
  • 98. Lan WJ, Ren HL, Pang Y, et al. (2012) A facile transport assay for H+ coupled membrane transport using fluorescence probes. Analytical Methods 4: 44–46.    
  • 99. Makui H, Roig E, Cole ST, et al. (2000) Identification of the Escherichia coli K-12 Nramp orthologue (MntH) as a selective divalent metal ion transporter. Mol Microbiol 35: 1065–1078.    
  • 100. Kehres DG, Zaharik ML, Finlay BB, et al. (2000) The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen. Mol Microbiol 36: 1085–1100.    
  • 101. Perry RD, Mier I, Fetherston JD (2007) Roles of the Yfe and Feo transporters of Yersinia pestis in iron uptake and intracellular growth. Biometals 20: 699–703.    
  • 102. Hohle TH, O'Brian MR (2009) The mntH gene encodes the major Mn(2+) transporter in Bradyrhizobium japonicum and is regulated by manganese via the Fur protein. Mol Microbiol 72: 399–409.    
  • 103. Fleming MD, Trenor CC, Su MA, et al. (1997) Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet 16: 383–386.
  • 104. Nevo Y, Nelson N (2004) The mutation F227I increases the coupling of metal ion transport in DCT1. J Biol Chem 279: 53056–53061.    
  • 105. Penmatsa A, Gouaux E (2014) How LeuT shapes our understanding of the mechanisms of sodium-coupled neurotransmitter transporters. J Physiol 592: 863–869.    
  • 106. Nevo Y (2008) Site-directed mutagenesis investigation of coupling properties of metal ion transport by DCT1. Biochim Biophys Acta 1778: 334–341.    
  • 107. Ivankov DN, Finkelstein AV, Kondrashov FA (2014) A structural perspective of compensatory evolution. Curr Opin Struct Biol 26: 104–112.    


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