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PMID: 19419962 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Substrate binding tunes conformational flexibility and kinetic stability of an amino acid antiporter.

The Journal of biological chemistry ·Vol. 284 ·No. 28 ·2009-07-10 ·Pages 18651-63

Bippes CA, Zeltina A, Casagrande F, Ratera M, Palacin M, Muller DJ, Fotiadis D

Abstract

We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state.

MeSH Terms
Amino Acids/chemistry Antiporters/chemistry,metabolism Bacillus subtilis/metabolism Bacterial Proteins/chemistry Binding Sites Biological Transport Biophysics/methods Cloning, Molecular Ion Transport Kinetics Molecular Conformation Substrate Specificity Thermodynamics
Chemicals
Amino Acids Antiporters Bacterial Proteins
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Bippes Christian A
Biotechnology Center, Technische Universität Dresden, D-01307 Dresden, Germany.
Zeltina Antra
Casagrande Fabio
Ratera Merce
Palacin Manuel
Muller Daniel J
Fotiadis Dimitrios
References (96)
96 references, click to expand
  1. AFM: a nanotool in membrane biology.
    Biochemistry. 2008 Aug 5;47(31):7986-98 PMID: 18616288
  2. Single protein misfolding events captured by atomic force microscopy.
    Nat Struct Biol. 1999 Nov;6(11):1025-8 PMID: 10542093
  3. Crystal structure of a glutamate/aspartate binding protein complexed with a glutamate molecule: structural basis of ligand specificity at atomic resolution.
    J Mol Biol. 2008 Sep 26;382(1):99-111 PMID: 18640128
  4. Transmembrane helices have rough energy surfaces.
    J Am Chem Soc. 2007 Jan 17;129(2):246-7 PMID: 17212383
  5. Redox imbalance in cystine/glutamate transporter-deficient mice.
    J Biol Chem. 2005 Nov 11;280(45):37423-9 PMID: 16144837
  6. Transducer binding establishes localized interactions to tune sensory rhodopsin II.
    Structure. 2008 Aug 6;16(8):1206-13 PMID: 18682222
  7. Dynamic energy landscape view of coupled binding and protein conformational change: induced-fit versus population-shift mechanisms.
    Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11182-7 PMID: 18678900
  8. Electromechanical limits of polymersomes.
    Phys Rev Lett. 2001 Nov 12;87(20):208301 PMID: 11690515
  9. Folding funnels, binding funnels, and protein function.
    Protein Sci. 1999 Jun;8(6):1181-90 PMID: 10386868
  10. Imaging and manipulation of biological structures with the AFM.
    Micron. 2002;33(4):385-97 PMID: 11814877
  11. Atomic force microscopy and spectroscopy of native membrane proteins.
    Nat Protoc. 2007;2(9):2191-7 PMID: 17853875
  12. Deciphering molecular interactions of native membrane proteins by single-molecule force spectroscopy.
    Annu Rev Biophys Biomol Struct. 2007;36:233-60 PMID: 17311527
  13. From Levinthal to pathways to funnels.
    Nat Struct Biol. 1997 Jan;4(1):10-9 PMID: 8989315
  14. Mechanical unfolding intermediates observed by single-molecule force spectroscopy in a fibronectin type III module.
    J Mol Biol. 2005 Jan 28;345(4):817-26 PMID: 15588828
  15. Anisotropic deformation response of single protein molecules.
    Proc Natl Acad Sci U S A. 2006 Aug 22;103(34):12724-8 PMID: 16908850
  16. Detecting molecular interactions that stabilize native bovine rhodopsin.
    J Mol Biol. 2006 Apr 21;358(1):255-69 PMID: 16519899
  17. Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters.
    Nature. 2005 Sep 8;437(7056):215-23 PMID: 16041361
  18. The PSIPRED protein structure prediction server.
    Bioinformatics. 2000 Apr;16(4):404-5 PMID: 10869041
  19. Projection structure of a member of the amino acid/polyamine/organocation transporter superfamily.
    J Biol Chem. 2008 Nov 28;283(48):33240-8 PMID: 18819925
  20. Identification and thermodynamic characterization of molten globule states of periplasmic binding proteins.
    Biochemistry. 2007 Sep 11;46(36):10339-52 PMID: 17696409
  21. Identification and characterisation of human xCT that co-expresses, with 4F2 heavy chain, the amino acid transport activity system xc-.
    Pflugers Arch. 2001 May;442(2):286-96 PMID: 11417227
  22. Exploring the energy landscape of GFP by single-molecule mechanical experiments.
    Proc Natl Acad Sci U S A. 2004 Nov 16;101(46):16192-7 PMID: 15531635
  23. Energy landscapes of biomolecular adhesion and receptor anchoring at interfaces explored with dynamic force spectroscopy.
    Faraday Discuss. 1998;(111):1-16 PMID: 10822596
  24. Examining the dynamic energy landscape of an antiporter upon inhibitor binding.
    J Mol Biol. 2008 Feb 1;375(5):1258-66 PMID: 18083192
  25. Probing the energy landscape of the membrane protein bacteriorhodopsin.
    Structure. 2004 May;12(5):871-9 PMID: 15130479
  26. Dynamic strength of molecular adhesion bonds.
    Biophys J. 1997 Apr;72(4):1541-55 PMID: 9083660
  27. Folding and binding cascades: dynamic landscapes and population shifts.
    Protein Sci. 2000 Jan;9(1):10-9 PMID: 10739242
  28. Functional and structural characterization of the first prokaryotic member of the L-amino acid transporter (LAT) family: a model for APC transporters.
    J Biol Chem. 2007 May 4;282(18):13270-81 PMID: 17344220
  29. Principles of protein folding--a perspective from simple exact models.
    Protein Sci. 1995 Apr;4(4):561-602 PMID: 7613459
  30. Ligand conduction and the gated-pore mechanism of transmembrane transport.
    Biochim Biophys Acta. 1997 Nov 21;1331(3):213-34 PMID: 9512653
  31. Cloning and expression of a plasma membrane cystine/glutamate exchange transporter composed of two distinct proteins.
    J Biol Chem. 1999 Apr 23;274(17):11455-8 PMID: 10206947
  32. The major amino acid transporter superfamily has a similar core structure as Na+-galactose and Na+-leucine transporters.
    Mol Membr Biol. 2008 Sep;25(6-7):567-70 PMID: 19031293
  33. Misfolding of membrane proteins in health and disease: the lady or the tiger?
    Curr Opin Struct Biol. 2000 Aug;10(4):438-42 PMID: 10981632
  34. Non-linear rate-equilibrium free energy relationships and Hammond behavior in protein folding.
    Biophys Chem. 2003;100(1-3):397-407 PMID: 12646379
  35. Stepwise unfolding of titin under force-clamp atomic force microscopy.
    Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):468-72 PMID: 11149943
  36. Entropic elasticity of lambda-phage DNA.
    Science. 1994 Sep 9;265(5178):1599-600 PMID: 8079175
  37. SLC7A7, encoding a putative permease-related protein, is mutated in patients with lysinuric protein intolerance.
    Nat Genet. 1999 Mar;21(3):297-301 PMID: 10080183
  38. Structure of a glutamate transporter homologue from Pyrococcus horikoshii.
    Nature. 2004 Oct 14;431(7010):811-8 PMID: 15483603
  39. Temperature softening of a protein in single-molecule experiments.
    J Mol Biol. 2005 Nov 25;354(2):497-503 PMID: 16246362
  40. Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter.
    Science. 2008 Oct 31;322(5902):709-13 PMID: 18927357
  41. Orientation of xanthophylls in phosphatidylcholine multibilayers.
    Biochim Biophys Acta. 1990 Apr 30;1023(3):405-12 PMID: 2334732
  42. Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy.
    J Mol Biol. 2004 Jul 23;340(5):1143-52 PMID: 15236973
  43. The experimental survey of protein-folding energy landscapes.
    Q Rev Biophys. 2005 Aug;38(3):245-88 PMID: 16780604
  44. Identification of SLC7A7, encoding y+LAT-1, as the lysinuric protein intolerance gene.
    Nat Genet. 1999 Mar;21(3):293-6 PMID: 10080182
  45. Fluctuations of primary ubiquitin folding intermediates in a force clamp.
    J Struct Biol. 2007 Mar;157(3):557-69 PMID: 17306561
  46. Discrete interactions in cell adhesion measured by single-molecule force spectroscopy.
    Nat Cell Biol. 2000 Jun;2(6):313-7 PMID: 10854320
  47. Hidden complexity in the mechanical properties of titin.
    Nature. 2003 Mar 27;422(6930):446-9 PMID: 12660787
  48. Atomic force microscopy reveals parallel mechanical unfolding pathways of T4 lysozyme: evidence for a kinetic partitioning mechanism.
    Proc Natl Acad Sci U S A. 2008 Feb 12;105(6):1885-90 PMID: 18272500
  49. Melittin-induced bilayer leakage depends on lipid material properties: evidence for toroidal pores.
    Biophys J. 2005 Mar;88(3):1828-37 PMID: 15596510
  50. New glycoprotein-associated amino acid transporters.
    J Membr Biol. 1999 Dec 1;172(3):181-92 PMID: 10568788
  51. Probing cellular microenvironments and tissue remodeling by atomic force microscopy.
    Pflugers Arch. 2008 Apr;456(1):29-49 PMID: 18058123
  52. Navigating the folding routes.
    Science. 1995 Mar 17;267(5204):1619-20 PMID: 7886447
  53. Ins and outs of major facilitator superfamily antiporters.
    Annu Rev Microbiol. 2008;62:289-305 PMID: 18537473
  54. Single-cell force spectroscopy.
    J Cell Sci. 2008 Jun 1;121(11):1785-91 PMID: 18492792
  55. Mechanism of Na+/H+ antiporting.
    Science. 2007 Aug 10;317(5839):799-803 PMID: 17690293
  56. Specific antigen/antibody interactions measured by force microscopy.
    Biophys J. 1996 May;70(5):2437-41 PMID: 9172770
  57. Essential aspartic acid residues, Asp-133, Asp-163 and Asp-164, in the transmembrane helices of a Na+/H+ antiporter (NhaA) from Escherichia coli.
    FEBS Lett. 1995 Apr 24;363(3):264-8 PMID: 7737413
  58. Structure and mechanism of ABC transporter proteins.
    Curr Opin Struct Biol. 2007 Aug;17(4):412-8 PMID: 17723295
  59. Models for the specific adhesion of cells to cells.
    Science. 1978 May 12;200(4342):618-27 PMID: 347575
  60. Alternating access and a pore-loop structure in the Na+-citrate transporter CitS of Klebsiella pneumoniae.
    J Biol Chem. 2004 Jul 23;279(30):31113-20 PMID: 15148311
  61. Direct measurement of the forces between complementary strands of DNA.
    Science. 1994 Nov 4;266(5186):771-3 PMID: 7973628
  62. Stretching single molecules into novel conformations using the atomic force microscope.
    Nat Struct Biol. 2000 Sep;7(9):719-24 PMID: 10966637
  63. Hammond behavior versus ground state effects in protein folding: evidence for narrow free energy barriers and residual structure in unfolded states.
    J Mol Biol. 2003 Apr 4;327(4):867-84 PMID: 12654269
  64. Application of physical organic chemistry to engineered mutants of proteins: Hammond postulate behavior in the transition state of protein folding.
    Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7814-8 PMID: 8356089
  65. Effect of chain length and unsaturation on elasticity of lipid bilayers.
    Biophys J. 2000 Jul;79(1):328-39 PMID: 10866959
  66. Point mutations in membrane proteins reshape energy landscape and populate different unfolding pathways.
    J Mol Biol. 2008 Feb 29;376(4):1076-90 PMID: 18191146
  67. Hydrodynamic effects in fast AFM single-molecule force measurements.
    Eur Biophys J. 2005 Feb;34(1):91-6 PMID: 15257425
  68. Mechanical properties of bovine rhodopsin and bacteriorhodopsin: possible roles in folding and function.
    Langmuir. 2008 Feb 19;24(4):1330-7 PMID: 18266338
  69. Detecting molecular interactions that stabilize, activate and guide ligand-binding of the sodium/proton antiporter MjNhaP1 from Methanococcus jannaschii.
    J Struct Biol. 2007 Aug;159(2):290-301 PMID: 17428680
  70. Dynamics of unfolded polypeptide chains as model for the earliest steps in protein folding.
    J Mol Biol. 2003 Sep 5;332(1):265-74 PMID: 12946363
  71. Neuroadaptations in cystine-glutamate exchange underlie cocaine relapse.
    Nat Neurosci. 2003 Jul;6(7):743-9 PMID: 12778052
  72. The genetics of heteromeric amino acid transporters.
    Physiology (Bethesda). 2005 Apr;20:112-24 PMID: 15772300
  73. Stability of bacteriorhodopsin alpha-helices and loops analyzed by single-molecule force spectroscopy.
    Biophys J. 2002 Dec;83(6):3578-88 PMID: 12496125
  74. Transport of amino acid-related compounds mediated by L-type amino acid transporter 1 (LAT1): insights into the mechanisms of substrate recognition.
    Mol Pharmacol. 2002 Apr;61(4):729-37 PMID: 11901210
  75. Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server.
    Nucleic Acids Res. 2007 Jul;35(Web Server issue):W429-32 PMID: 17483518
  76. Identification of the cadaverine recognition site on the cadaverine-lysine antiporter CadB.
    J Biol Chem. 2006 Sep 29;281(39):29213-20 PMID: 16877381
  77. Reversible unfolding of individual titin immunoglobulin domains by AFM.
    Science. 1997 May 16;276(5315):1109-12 PMID: 9148804
  78. Adhesion forces between individual ligand-receptor pairs.
    Science. 1994 Apr 15;264(5157):415-7 PMID: 8153628
  79. The speed limit for protein folding measured by triplet-triplet energy transfer.
    Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9597-601 PMID: 10449738
  80. Atomic force microscope.
    Phys Rev Lett. 1986 Mar 3;56(9):930-933 PMID: 10033323
  81. Cystinuria caused by mutations in rBAT, a gene involved in the transport of cystine.
    Nat Genet. 1994 Apr;6(4):420-5 PMID: 8054986
  82. Flexibility in the ABC transporter MsbA: Alternating access with a twist.
    Proc Natl Acad Sci U S A. 2007 Nov 27;104(48):19005-10 PMID: 18024585
  83. High-throughput single-molecule force spectroscopy for membrane proteins.
    Nanotechnology. 2008 Sep 24;19(38):384014 PMID: 21832573
  84. The amino acid/polyamine/organocation (APC) superfamily of transporters specific for amino acids, polyamines and organocations.
    Microbiology (Reading). 2000 Aug;146 ( Pt 8):1797-1814 PMID: 10931886
  85. Amino acid transporters ASCT2 and LAT1 in cancer: partners in crime?
    Semin Cancer Biol. 2005 Aug;15(4):254-66 PMID: 15916903
  86. Unfolding pathways of individual bacteriorhodopsins.
    Science. 2000 Apr 7;288(5463):143-6 PMID: 10753119
  87. Structure and mechanics of membrane proteins.
    Annu Rev Biochem. 2008;77:127-48 PMID: 18518819
  88. Probing origins of molecular interactions stabilizing the membrane proteins halorhodopsin and bacteriorhodopsin.
    Structure. 2005 Feb;13(2):235-42 PMID: 15698567
  89. Role of extracellular glutamic acids in the stability and energy landscape of bacteriorhodopsin.
    Biophys J. 2008 Oct;95(7):3407-18 PMID: 18621827
  90. Locating ligand binding and activation of a single antiporter.
    EMBO Rep. 2005 Jul;6(7):668-74 PMID: 15962009
  91. Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT.
    Nat Genet. 1999 Sep;23(1):52-7 PMID: 10471498
  92. Unfolding barriers in bacteriorhodopsin probed from the cytoplasmic and the extracellular side by AFM.
    Structure. 2006 Mar;14(3):521-7 PMID: 16531236
  93. Mechanical and chemical unfolding of a single protein: a comparison.
    Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3694-9 PMID: 10097099
  94. Folding and assembly of proteorhodopsin.
    J Mol Biol. 2008 Feb 8;376(1):35-41 PMID: 18155728
  95. Membrane topology of system xc- light subunit reveals a re-entrant loop with substrate-restricted accessibility.
    J Biol Chem. 2004 Jul 23;279(30):31228-36 PMID: 15151999
  96. Aminosulfonate modulated pH-induced conformational changes in connexin26 hemichannels.
    J Biol Chem. 2007 Mar 23;282(12):8895-904 PMID: 17227765
Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
0021-9258
DOI
10.1074/jbc.M109.004267
Published
2009-07-10
Epub
2009-00-06
Pages
18651-63
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC2707244
Subset
IM
External Links
PubMed source DOI: 10.1074/jbc.M109.004267 PMC Full Text View journal details
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