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Structural Flexibility of Tau in Its Interaction with Microtubules as Viewed by Site-Directed Spin Labeling EPR Spectroscopy

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Tau Protein

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2754))

Abstract

Tau is a microtubule-associated protein that belongs to the Intrinsically Disordered Proteins (IDPs) family. IDPs or Intrinsically Disordered Regions (IDRs) play key roles in protein interaction networks and their dysfunctions are often related to severe diseases. Defined by their lack of stable secondary and tertiary structures in physiological conditions while being functional, these proteins use their inherent structural flexibility to adapt to and interact with various binding partners. Knowledges on the structural dynamics of IDPs and their different conformers are crucial to finely decipher fundamental biological processes controlled by mechanisms such as conformational adaptations or switches, induced fit, or conformational selection events. Different mechanisms of binding have been proposed: among them, the so-called folding-upon-binding in which the IDP adopts a certain conformation upon interacting with a partner protein, or the formation of a “fuzzy” complex in which the IDP partly keeps its dynamical character at the surface of its partner. The dynamical nature and physicochemical properties of unbound as well as bound IDPs make this class of proteins particularly difficult to characterize by classical bio-structural techniques and require specific approaches for the fine description of their inherent dynamics.

Among other techniques, Site-Directed Spin Labeling combined with Electron Paramagnetic Resonance (SDSL-EPR) spectroscopy has gained much interest in this last decade for the study of IDPs. SDSL-EPR consists in grafting a paramagnetic label (mainly a nitroxide radical) at selected site(s) of the macromolecule under interest followed by its observation using and/or combining different EPR strategies. These nitroxide spin labels detected by continuous wave (cw) EPR spectroscopy are used as perfect reporters or “spy spins” of their local environment, being able to reveal structural transitions, folding/unfolding events, etc. Another approach is based on the measurement of inter-label distance distributions in the 1.5–8.0 nm range using pulsed dipolar EPR experiments, such as Double Electron-Electron Resonance (DEER) spectroscopy. The technique is then particularly well suited to study the behavior of Tau in its interaction with its physiological partner: microtubules (MTs). In this chapter we provide a detailed experimental protocol for the labeling of Tau protein and its EPR study while interacting with preformed (Paclitaxel-stabilized) MTs, or using Tau as MT inducer. We show how the choice of nitroxide label can be crucial to obtain functional information on Tau/tubulin complexes.

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References

  1. Fuxreiter M (2012) Fuzziness: linking regulation to protein dynamics. Mol BioSyst 8:168–177. https://doi.org/10.1039/C1MB05234A

    Article  CAS  PubMed  Google Scholar 

  2. Dyson HJ, Wright PE (2005) Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol 6:197–208. https://doi.org/10.1038/nrm1589

    Article  CAS  PubMed  Google Scholar 

  3. Babu MM, van der Lee R, de Groot NS, Gsponer J (2011) Intrinsically disordered proteins: regulation and disease. Curr Opin Struct Biol 21:432–440. https://doi.org/10.1016/j.sbi.2011.03.011

    Article  CAS  PubMed  Google Scholar 

  4. Brion J-P, Passareiro H, Nunez J, Flament-Durand J (1985) Mise en évidence immunologique de la protéine tau au niveau des lésions de dégénérescence neurofibrillaire de la maladie d’Alzheimer; Alzheimer’s disease: immunocytochemical detection of tau protein in neurofibrillary tangles. Arch Biol 96:229–235

    CAS  Google Scholar 

  5. Goode BL, Denis PE, Panda D, Radeke MJ, Miller HP, Wilson L, Feinstein SC (1997) Functional interactions between the proline-rich and repeat regions of tau enhance microtubule binding and assembly. Mol Biol Cell 8:353–365. https://doi.org/10.1091/mbc.8.2.353

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Preuss U, Biernat J, Mandelkow EM, Mandelkow E (1997) The “jaws” model of tau-microtubule interaction examined in CHO cells. J Cell Sci 110(Pt 6):789–800. https://doi.org/10.1242/jcs.110.6.789

    Article  CAS  PubMed  Google Scholar 

  7. Mukrasch MD, von Bergen M, Biernat J, Fischer D, Griesinger C, Mandelkow E, Zweckstetter M (2007) The “jaws” of the tau-microtubule interaction. J Biol Chem 282:12230–12239. https://doi.org/10.1074/jbc.M607159200

    Article  CAS  PubMed  Google Scholar 

  8. Sillen A, Barbier P, Landrieu I, Lefebvre S, Wieruszeski J-M, Leroy A, Peyrot V, Lippens G (2007) NMR investigation of the interaction between the neuronal protein tau and the microtubules. Biochemistry 46:3055–3064. https://doi.org/10.1021/bi061920i

    Article  CAS  PubMed  Google Scholar 

  9. Kar S, Fan J, Smith MJ, Goedert M, Amos LA (2003) Repeat motifs of tau bind to the insides of microtubules in the absence of taxol. EMBO J 22:70–77. https://doi.org/10.1093/emboj/cdg001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Nogales E, Wolf SG, Downing KH (1998) Structure of the alpha beta tubulin dimer by electron crystallography. Nature 391:199–203. https://doi.org/10.1038/34465

    Article  CAS  PubMed  Google Scholar 

  11. Santarella RA, Skiniotis G, Goldie KN, Tittmann P, Gross H, Mandelkow E-M, Mandelkow E, Hoenger A (2004) Surface-decoration of microtubules by human tau. J Mol Biol 339:539–553. https://doi.org/10.1016/j.jmb.2004.04.008

    Article  CAS  PubMed  Google Scholar 

  12. Tsvetkov PO, Makarov AA, Malesinski S, Peyrot V, Devred F (2012) New insights into tau–microtubules interaction revealed by isothermal titration calorimetry. Biochimie 94:916–919. https://doi.org/10.1016/j.biochi.2011.09.011

    Article  CAS  PubMed  Google Scholar 

  13. Gigant B, Landrieu I, Fauquant C, Barbier P, Huvent I, Wieruszeski J-M, Knossow M, Lippens G (2014) Mechanism of Tau-promoted microtubule assembly as probed by NMR spectroscopy. J Am Chem Soc 136:12615–12623. https://doi.org/10.1021/ja504864m

    Article  CAS  PubMed  Google Scholar 

  14. Kellogg EH, Hejab NMA, Poepsel S, Downing KH, DiMaio F, Nogales E (2018) Near-atomic model of microtubule-tau interactions. Science 360:1242–1246. https://doi.org/10.1126/science.aat1780

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Martinho M, Allegro D, Huvent I, Chabaud C, Etienne E, Kovacic H, Guigliarelli B, Peyrot V, Landrieu I, Belle V, Barbier P (2018) Two Tau binding sites on tubulin revealed by thiol-disulfide exchanges. Sci Rep 8:13846. https://doi.org/10.1038/s41598-018-32096-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Martinho M, Habchi J, El Habre Z, Nesme L, Guigliarelli B, Belle V, Longhi S (2013) Assessing induced folding within the intrinsically disordered C-terminal domain of the Henipavirus nucleoproteins by site-directed spin labeling EPR spectroscopy. J Biomol Struct Dyn 31:453–471. https://doi.org/10.1080/07391102.2012.706068

    Article  CAS  PubMed  Google Scholar 

  17. Le Breton N, Martinho M, Mileo E, Etienne E, Gerbaud G, Guigliarelli B, Belle V (2015) Exploring intrinsically disordered proteins using site-directed spin labeling electron paramagnetic resonance spectroscopy. Front Mol Biosci 2. https://doi.org/10.3389/fmolb.2015.00021

  18. Habchi J, Martinho M, Gruet A, Guigliarelli B, Longhi S, Belle V (2012) Monitoring structural transitions in IDPs by site-directed spin labeling EPR spectroscopy. In: Uversky VN, Dunker AK (eds) Intrinsically disordered protein analysis. Humana Press, Totowa, pp 361–386

    Chapter  Google Scholar 

  19. Pierro A, Etienne E, Gerbaud G, Guigliarelli B, Ciurli S, Belle V, Zambelli B, Mileo E (2020) Nickel and GTP modulate helicobacter pylori UreG structural flexibility. Biomol Ther 10. https://doi.org/10.3390/biom10071062

  20. Martinho M, Fournier E, Le Breton N, Mileo E, Belle V (2018) Nitroxide spin labels: fabulous spy spins for biostructural EPR applications. In: Chechik V, Murphy DM (eds) Electron paramagnetic resonance. Royal Society of Chemistry, Cambridge, pp 66–88

    Chapter  Google Scholar 

  21. Torricella F, Pierro A, Mileo E, Belle V, Bonucci A (1869) Nitroxide spin labels and EPR spectroscopy: a powerful association for protein dynamics studies. Biochim Biophys Acta - Proteins and Proteomics 2021:140653. https://doi.org/10.1016/j.bbapap.2021.140653

    Article  CAS  Google Scholar 

  22. Klare JP (2013) Site-directed spin labeling EPR spectroscopy in protein research. Biol Chem 394:1281–1300. https://doi.org/10.1515/hsz-2013-0155

    Article  CAS  PubMed  Google Scholar 

  23. Drescher M (2011) EPR in protein science. In: Drescher M, Jeschke G (eds) EPR spectroscopy. Springer Berlin Heidelberg, Berlin, Heidelberg, pp 91–119

    Chapter  Google Scholar 

  24. Klare JP (2017) Site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy: a versatile tool to study protein-protein interactions. Protein Interactions 22

    Google Scholar 

  25. Hubbell WL, Cafiso DS, Altenbach C (2000) Identifying conformational changes with site-directed spin labeling. Nat Struct Biol 7:5

    Article  Google Scholar 

  26. Morin B, Bourhis J-M, Belle V, Woudstra M, Carrière F, Guigliarelli B, Fournel A, Longhi S (2006) Assessing induced folding of an intrinsically disordered protein by site-directed spin-labeling electron paramagnetic resonance spectroscopy. J Phys Chem B 110:20596–20608. https://doi.org/10.1021/jp063708u

    Article  CAS  PubMed  Google Scholar 

  27. Belle V, Rouger S, Costanzo S, Liquière E, Strancar J, Guigliarelli B, Fournel A, Longhi S (2008) Map** α-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy. Proteins 73:973–988. https://doi.org/10.1002/prot.22125

    Article  CAS  PubMed  Google Scholar 

  28. Margittai M, Langen R (2006) Spin labeling analysis of amyloids and other protein aggregates. In: Methods in enzymology. Elsevier, pp 122–139

    Google Scholar 

  29. Meyer V, Margittai M (2016) Spin labeling and characterization of tau fibrils using electron paramagnetic resonance (EPR). In: Eliezer D (ed) Protein amyloid aggregation. Springer New York, New York, pp 185–199

    Chapter  Google Scholar 

  30. Török M, Milton S, Kayed R, Wu P, McIntire T, Glabe CG, Langen R (2002) Structural and dynamic features of Alzheimer’s Aβ peptide in amyloid fibrils studied by site-directed spin labeling. J Biol Chem 277:40810–40815. https://doi.org/10.1074/jbc.M205659200

    Article  CAS  PubMed  Google Scholar 

  31. Le Breton N, Longhi S, Rockenbauer A, Guigliarelli B, Marque SRA, Belle V, Martinho M (2019) Probing the dynamic properties of two sites simultaneously in a protein–protein interaction process: a SDSL-EPR study. Phys Chem Chem Phys 21:22584–22588. https://doi.org/10.1039/C9CP04660G

    Article  PubMed  Google Scholar 

  32. Jeschke G, Wegener C, Nietschke M, Jung H, Steinhoff H-J (2004) Interresidual distance determination by four-pulse double electron-electron resonance in an integral membrane protein: the Na+/Proline transporter PutP of Escherichia coli. Biophys J 86:2551–2557. https://doi.org/10.1016/S0006-3495(04)74310-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Jeschke G (2018) The contribution of modern EPR to structural biology. Emerg Top Life Sci 2:9–18. https://doi.org/10.1042/ETLS20170143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Fournier E, Tachon S, Fowler NJ, Gerbaud G, Mansuelle P, Dorlet P, Visser SP, Belle V, Simaan AJ, Martinho M (2019) The hunt for the closed conformation of the fruit-ripening enzyme 1-aminocyclopropane-1-carboxylic oxidase: a combined electron paramagnetic resonance and molecular dynamics study. Chem Eur J 25:13766–13776. https://doi.org/10.1002/chem.201903003

    Article  CAS  PubMed  Google Scholar 

  35. Pannier M, Veit S, Godt A, Jeschke G, Spiess HW (2011) Dead-time free measurement of dipole-dipole interactions between electron spins. 2000. J Magn Reson 213:316–325. https://doi.org/10.1016/j.jmr.2011.08.035

    Article  CAS  PubMed  Google Scholar 

  36. Zeng Z, Fichou Y, Vigers M, Tsay K, Han S (2022) Illuminating the structural basis of tau aggregation by intramolecular distance tracking: a perspective on methods. J Phys Chem B 126:6384–6395. https://doi.org/10.1021/acs.jpcb.2c02022

    Article  CAS  PubMed  Google Scholar 

  37. Fichou Y, Eschmann NA, Keller TJ, Han S (2017) Conformation-based assay of tau protein aggregation. Methods Cell Biol 141:89–112. https://doi.org/10.1016/bs.mcb.2017.06.008

    Article  CAS  PubMed  Google Scholar 

  38. Klare JP, Steinhoff H-J (2009) Spin labeling EPR. Photosynth Res 102:377–390. https://doi.org/10.1007/s11120-009-9490-7

    Article  CAS  PubMed  Google Scholar 

  39. Pirman NL, Milshteyn E, Galiano L, Hewlett JC, Fanucci GE (2011) Characterization of the disordered-to-α-helical transition of IA 3 by SDSL-EPR spectroscopy: characterization of IA 3 by SDSL-EPR. Protein Sci 20:150–159. https://doi.org/10.1002/pro.547

    Article  CAS  PubMed  Google Scholar 

  40. Etienne E, Le Breton N, Martinho M, Mileo E, Belle V (2017) SimLabel: a graphical user interface to simulate continuous wave EPR spectra from site-directed spin labeling experiments: multi-components SDSL-EPR spectra simulation using SimLabel. Magn Reson Chem 55:714–719. https://doi.org/10.1002/mrc.4578

    Article  CAS  PubMed  Google Scholar 

  41. Stoll S, Schweiger A (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J Magn Reson 178:42–55. https://doi.org/10.1016/j.jmr.2005.08.013

    Article  CAS  PubMed  Google Scholar 

  42. Mileo E, Lorenzi M, Erales J, Lignon S, Puppo C, Le Breton N, Etienne E, Marque SRA, Guigliarelli B, Gontero B, Belle V (2013) Dynamics of the intrinsically disordered protein CP12 in its association with GAPDH in the green alga Chlamydomonas reinhardtii: a fuzzy complex. Mol BioSyst 9:2869. https://doi.org/10.1039/c3mb70190e

    Article  CAS  PubMed  Google Scholar 

  43. Qu K, Vaughn JL, Sienkiewicz A, Scholes CP, Fetrow JS (1997) Kinetics and motional dynamics of spin-labeled yeast iso-1-cytochrome c: 1. Stopped-flow electron paramagnetic resonance as a probe for protein folding/unfolding of the C-terminal helix spin-labeled at cysteine 102. Biochemistry 36:2884–2897. https://doi.org/10.1021/bi962155i

    Article  CAS  PubMed  Google Scholar 

  44. Goldman SA, Bruno GV, Polnaszek CF, Freed JH (1972) An ESR study of anisotropic rotational reorientation and slow tumbling in liquid and frozen media. J Chem Phys 56:716–735. https://doi.org/10.1063/1.1677222

    Article  CAS  Google Scholar 

  45. Alushin GM, Lander GC, Kellogg EH, Zhang R, Baker D, Nogales E (2014) High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis. Cell 157:1117–1129. https://doi.org/10.1016/j.cell.2014.03.053

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. De Bessa T, Breuzard G, Allegro D, Devred F, Peyrot V, Barbier P (2017) Tau interaction with tubulin and microtubules: from purified proteins to cells. Methods Mol Biol 1523:61–85. https://doi.org/10.1007/978-1-4939-6598-4_4

    Article  CAS  PubMed  Google Scholar 

  47. Gaskin F, Cantor CR, Shelanski ML (1975) Biochemical studies on the in vitro assembly and disassembly of microtubules. Ann N Y Acad Sci 253:133–146. https://doi.org/10.1111/j.1749-6632.1975.tb19197.x

    Article  CAS  PubMed  Google Scholar 

  48. Getz EB, **ao M, Chakrabarty T, Cooke R, Selvin PR (1999) A comparison between the sulfhydryl reductants Tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal Biochem 273:73–80. https://doi.org/10.1006/abio.1999.4203

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the Agence Nationale de la Recherche (ANR-21-CE29-0024 MAGNETAU), the Centre National de la Recherche Scientifique (CNRS), and Aix-Marseille Université (AMU). Financial support from the IR INFRANALYTICS FR2054 for conducting the research is gratefully acknowledged.

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Authors and Affiliations

  1. Aix Marseille Univ, CNRS, BIP, Marseille, France

    Marlène Martinho, Emilien Etienne, Alessio Bonucci & Valérie Belle

  2. Aix Marseille Univ, CNRS, INP, Inst Neurophysiopathol, Marseille, France

    Diane Allegro, Cynthia Lohberger & Pascale Barbier

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  1. Marlène Martinho
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  2. Diane Allegro
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  3. Emilien Etienne
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  4. Cynthia Lohberger
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  5. Alessio Bonucci
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  6. Valérie Belle
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  7. Pascale Barbier
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Correspondence to Marlène Martinho or Pascale Barbier .

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  1. Inserm U1167, University of Lille, Villeneuve d’Ascq Cedex, France

    Caroline Smet-Nocca

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Martinho, M. et al. (2024). Structural Flexibility of Tau in Its Interaction with Microtubules as Viewed by Site-Directed Spin Labeling EPR Spectroscopy. In: Smet-Nocca, C. (eds) Tau Protein. Methods in Molecular Biology, vol 2754. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3629-9_3

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  • DOI: https://doi.org/10.1007/978-1-0716-3629-9_3

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