Nuclear magnetic resonance (NMR) spectroscopy has been instrumental during the past two decades in providing high-resolution structures of protein complexes. It has been the method of choice for determining the structure of dynamic protein complexes, which are typically recalcitrant to other structural techniques. Until recently, NMR spectroscopy has yielded structures of small or medium-sized protein complexes, up to approximately 30–40 kDa. Major breakthroughs during the past decade, especially in isotope-labeling techniques, have enabled NMR characterization of large protein systems with molecular weights of hundreds of kDa. This has provided unique insights into the binding, dynamic, and allosteric properties of large systems. Notably, there is now a slowly but steadily growing list of large, dynamic protein complexes whose atomic structure has been determined by NMR. Many of these complexes are characterized by a high degree of flexibility and, thus, their structures could not have been obtained using other structural methods. Especially in the field of molecular chaperones, NMR has recently provided the first-ever high-resolution structures of their complexes with unfolded proteins. Further technological advances will establish NMR as the primary tool for obtaining atomic structures of challenging systems with even higher complexity.


Article metrics loading...

Loading full text...

Full text loading...


Literature Cited

  1. Ahnert SE, Marsh JA, Hernandez H, Robinson CV, Teichmann SA. 1.  2015. Principles of assembly reveal a periodic table of protein complexes. Science 350:aaa2245 [Google Scholar]
  2. Aittaleb M, Rashid R, Chen Q, Palmer JR, Daniels CJ, Li H. 2.  2003. Structure and function of archaeal box C/D sRNP core proteins. Nat. Struct. Mol. Biol. 10:256–63 [Google Scholar]
  3. Ali MM, Roe SM, Vaughan CK, Meyer P, Panaretou B. 3.  et al. 2006. Crystal structure of an Hsp90–nucleotide–p23/Sba1 closed chaperone complex. Nature 440:1013–17 [Google Scholar]
  4. Bechtluft P, Nouwen N, Tans SJ, Driessen AJ. 4.  2010. SecB—a chaperone dedicated to protein translocation. Mol. Biosyst. 6:620–27 [Google Scholar]
  5. Becker PB, Horz W. 5.  2002. ATP-dependent nucleosome remodeling. Annu. Rev. Biochem. 71:247–73 [Google Scholar]
  6. Boehr DD, Nussinov R, Wright PE. 6.  2009. The role of dynamic conformational ensembles in biomolecular recognition. Nat. Chem. Biol. 5:789–96 [Google Scholar]
  7. Bukau B, Weissman J, Horwich A. 7.  2006. Molecular chaperones and protein quality control. Cell 125:443–51 [Google Scholar]
  8. Chatzi KE, Sardis MF, Economou A, Karamanou S. 8.  2014. SecA-mediated targeting and translocation of secretory proteins. Biochim. Biophys. Acta 1843:1466–74 [Google Scholar]
  9. Chen L, Balabanidou V, Remeta DP, Minetti CA, Portaliou AG. 9.  et al. 2011. Structural instability tuning as a regulatory mechanism in protein–protein interactions. Mol. Cell 44:734–44 [Google Scholar]
  10. Clore GM. 10.  2015. Practical aspects of paramagnetic relaxation enhancement in biological macromolecules. Methods Enzymol 564:485–97 [Google Scholar]
  11. Dollins DE, Warren JJ, Immormino RM, Gewirth DT. 11.  2007. Structures of GRP94–nucleotide complexes reveal mechanistic differences between the hsp90 chaperones. Mol. Cell 28:41–56 [Google Scholar]
  12. Dominguez C, Boelens R, Bonvin AM. 12.  2003. HADDOCK: a protein–protein docking approach based on biochemical or biophysical information. J. Am. Chem. Soc. 125:1731–37 [Google Scholar]
  13. Ferbitz L, Maier T, Patzelt H, Bukau B, Deuerling E, Ban N. 13.  2004. Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins. Nature 431:590–96 [Google Scholar]
  14. Gelis I, Bonvin AM, Keramisanou D, Koukaki M, Gouridis G. 14.  et al. 2007. Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR. Cell 131:756–69 [Google Scholar]
  15. Genevaux P, Keppel F, Schwager F, Langendijk-Genevaux PS, Hartl FU, Georgopoulos C. 15.  2004. In vivo analysis of the overlapping functions of DnaK and trigger factor. EMBO Rep 5:195–200 [Google Scholar]
  16. Gouridis G, Karamanou S, Gelis I, Kalodimos CG, Economou A. 16.  2009. Signal peptides are allosteric activators of the protein translocase. Nature 462:363–67 [Google Scholar]
  17. Harrison CJ, Hayer-Hartl M, Di Liberto M, Hartl F, Kuriyan J. 17.  1997. Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK. Science 276:431–35 [Google Scholar]
  18. Haslberger T, Zdanowicz A, Brand I, Kirstein J, Turgay K. 18.  et al. 2008. Protein disaggregation by the AAA+ chaperone ClpB involves partial threading of looped polypeptide segments. Nat. Struct. Mol. Biol. 15:641–50 [Google Scholar]
  19. Hiller S, Wagner G. 19.  2009. The role of solution NMR in the structure determinations of VDAC-1 and other membrane proteins. Curr. Opin. Struct. Biol. 19:396–401 [Google Scholar]
  20. Hock R, Furusawa T, Ueda T, Bustin M. 20.  2007. HMG chromosomal proteins in development and disease. Trends Cell Biol 17:72–79 [Google Scholar]
  21. Huang C, Rossi P, Saio T, Kalodimos CG. 21.  2016. Structural basis for the antifolding activity of a molecular chaperone. Nature 537:202–6 [Google Scholar]
  22. Imai S, Osawa M, Takeuchi K, Shimada I. 22.  2010. Structural basis underlying the dual gate properties of KcsA. PNAS 107:6216–21 [Google Scholar]
  23. Ittner LM, Ke YD, Delerue F, Bi M, Gladbach A. 23.  et al. 2010. Dendritic function of tau mediates amyloid-β toxicity in Alzheimer's disease mouse models. Cell 142:387–97 [Google Scholar]
  24. Kainosho M, Torizawa T, Iwashita Y, Terauchi T, Mei Ono A, Güntert P. 24.  2006. Optimal isotope labelling for NMR protein structure determinations. Nature 440:52–57 [Google Scholar]
  25. Kaiser CM, Chang HC, Agashe VR, Lakshmipathy SK, Etchells SA. 25.  et al. 2006. Real-time observation of trigger factor function on translating ribosomes. Nature 444:455–60 [Google Scholar]
  26. Kalodimos CG. 26.  2012. Protein function and allostery: a dynamic relationship. Ann. N.Y. Acad. Sci. 1260:81–86 [Google Scholar]
  27. Karagöz GE, Duarte AM, Akoury E, Ippel H, Biernat J. 27.  et al. 2014. Hsp90–Tau complex reveals molecular basis for specificity in chaperone action. Cell 156:963–74 [Google Scholar]
  28. Karagöz GE, Duarte AM, Ippel H, Uetrecht C, Sinnige T. 28.  et al. 2011. N-terminal domain of human Hsp90 triggers binding to the cochaperone p23. PNAS 108:580–85 [Google Scholar]
  29. Kasinath V, Valentine KG, Wand AJ. 29.  2013. A 13C labeling strategy reveals a range of aromatic side chain motion in calmodulin. J. Am. Chem. Soc. 135:9560–63 [Google Scholar]
  30. Kato H, van Ingen H, Zhou BR, Feng H, Bustin M. 30.  et al. 2011. Architecture of the high mobility group nucleosomal protein 2–nucleosome complex as revealed by methyl-based NMR. PNAS 108:12283–88 [Google Scholar]
  31. Kay LE. 31.  2011. Solution NMR spectroscopy of supra-molecular systems, why bother? A methyl-TROSY view. J. Magn. Reson. 210:159–70 [Google Scholar]
  32. Kerfah R, Plevin MJ, Sounier R, Gans P, Boisbouvier J. 32.  2015. Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins. Curr. Opin. Struct. Biol. 32:113–22 [Google Scholar]
  33. Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Hartl FU. 33.  2013. Molecular chaperone functions in protein folding and proteostasis. Annu. Rev. Biochem. 82:323–55 [Google Scholar]
  34. Krukenberg KA, Forster F, Rice LM, Sali A, Agard DA. 34.  2008. Multiple conformations of E.coli Hsp90 in solution: insights into the conformational dynamics of Hsp90. Structure 16:755–65 [Google Scholar]
  35. Lapinaite A, Simon B, Skjaerven L, Rakwalska-Bange M, Gabel F, Carlomagno T. 35.  2013. The structure of the box C/D enzyme reveals regulation of RNA methylation. Nature 502:519–23 [Google Scholar]
  36. Latham MP, Sekhar A, Kay LE. 36.  2014. Understanding the mechanism of proteasome 20S core particle gating. PNAS 111:5532–37 [Google Scholar]
  37. Li J, Buchner J. 37.  2013. Structure, function and regulation of the Hsp90 machinery. Biomed. J 36106–17 [Google Scholar]
  38. Lin J, Lai S, Jia R, Xu A, Zhang L. 38.  et al. 2011. Structural basis for site-specific ribose methylation by box C/D RNA protein complexes. Nature 469:559–63 [Google Scholar]
  39. Ma C, Li W, Xu Y, Rizo J. 39.  2011. Munc13 mediates the transition from the closed syntaxin–Munc18 complex to the SNARE complex. Nat. Struct. Mol. Biol. 18:542–49 [Google Scholar]
  40. Marsh JA, Teichmann SA. 40.  2015. Structure, dynamics, assembly, and evolution of protein complexes. Annu. Rev. Biochem. 84:551–75 [Google Scholar]
  41. Marsh JA, Teichmann SA, Forman-Kay JD. 41.  2012. Probing the diverse landscape of protein flexibility and binding. Curr. Opin. Struct. Biol. 22:643–50 [Google Scholar]
  42. Mattoo RU, Goloubinoff P. 42.  2014. Molecular chaperones are nanomachines that catalytically unfold misfolded and alternatively folded proteins. Cell. Mol. Life Sci. 71:3311–25 [Google Scholar]
  43. Milbradt AG, Arthanari H, Takeuchi K, Boeszoermenyi A, Hagn F, Wagner G. 43.  2015. Increased resolution of aromatic cross peaks using alternate 13C labeling and TROSY. J. Biomol. NMR 62:291–301 [Google Scholar]
  44. Mittag T, Kay LE, Forman-Kay JD. 44.  2010. Protein dynamics and conformational disorder in molecular recognition. J. Mol. Recognit. 23:105–16 [Google Scholar]
  45. Monneau YR, Ishida Y, Rossi P, Saio T, Tzeng SR. 45.  et al. 2016. Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications. J. Biomol. NMR 65:99–108 [Google Scholar]
  46. Mund M, Overbeck JH, Ullmann J, Sprangers R. 46.  2013. LEGO-NMR spectroscopy: a method to visualize individual subunits in large heteromeric complexes. Angew. Chem. Int. Ed. Engl. 52:11401–5 [Google Scholar]
  47. Nooren IM, Thornton JM. 47.  2003. Diversity of protein–protein interactions. EMBO J 22:3486–92 [Google Scholar]
  48. Oh E, Becker AH, Sandikci A, Huber D, Chaba R. 48.  et al. 2011. Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo. Cell 147:1295–308 [Google Scholar]
  49. Papanikolau Y, Papadovasilaki M, Ravelli RB, McCarthy AA, Cusack S. 49.  et al. 2007. Structure of dimeric SecA, the Escherichia coli preprotein translocase motor. J. Mol. Biol. 366:1545–57 [Google Scholar]
  50. Papanikou E, Karamanou S, Economou A. 50.  2007. Bacterial protein secretion through the translocase nanomachine. Nat. Rev. Microbiol. 5:839–51 [Google Scholar]
  51. Parsell DA, Kowal AS, Singer MA, Lindquist S. 51.  1994. Protein disaggregation mediated by heat-shock protein Hsp104. Nature 372:475–78 [Google Scholar]
  52. Pearl LH. 52.  2016. The HSP90 molecular chaperone—an enigmatic ATPase. Biopolymers 105:594–607 [Google Scholar]
  53. Pearl LH, Prodromou C. 53.  2006. Structure and mechanism of the Hsp90 molecular chaperone machinery. Annu. Rev. Biochem. 75:271–94 [Google Scholar]
  54. Popovych N, Tzeng SR, Tonelli M, Ebright RH, Kalodimos CG. 54.  2009. Structural basis for cAMP-mediated allosteric control of the catabolite activator protein. PNAS 106:6927–32 [Google Scholar]
  55. Rapoport TA. 55.  2007. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature 450:663–69 [Google Scholar]
  56. Rosenzweig R, Kay LE. 56.  2014. Bringing dynamic molecular machines into focus by methyl-TROSY NMR. Annu. Rev. Biochem. 83:291–315 [Google Scholar]
  57. Rosenzweig R, Moradi S, Zarrine-Afsar A, Glover JR, Kay LE. 57.  2013. Unraveling the mechanism of protein disaggregation through a ClpB–DnaK interaction. Science 339:1080–83 [Google Scholar]
  58. Ruschak AM, Religa TL, Breuer S, Witt S, Kay LE. 58.  2010. The proteasome antechamber maintains substrates in an unfolded state. Nature 467:868–71 [Google Scholar]
  59. Russel D, Lasker K, Webb B, Velazquez-Muriel J, Tjioe E. 59.  et al. 2012. Putting the pieces together: integrative modeling platform software for structure determination of macromolecular assemblies. PLOS Biol 10:e1001244 [Google Scholar]
  60. Saibil HR. 60.  2008. Chaperone machines in action. Curr. Opin. Struct. Biol. 18:35–42 [Google Scholar]
  61. Saibil HR. 61.  2013. Chaperone machines for protein folding, unfolding and disaggregation. Nat. Rev Mol. Cell Biol. 14:630–42 [Google Scholar]
  62. Saio T, Guan X, Rossi P, Economou A, Kalodimos CG. 62.  2014. Structural basis for protein antiaggregation activity of the trigger factor chaperone. Science 344:1250494 [Google Scholar]
  63. Sala A, Bordes P, Genevaux P. 63.  2014. Multitasking SecB chaperones in bacteria. Front. Microbiol. 5:666 [Google Scholar]
  64. Sprangers R, Kay LE. 64.  2007. Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 445:618–22 [Google Scholar]
  65. Street TO, Lavery LA, Agard DA. 65.  2011. Substrate binding drives large-scale conformational changes in the Hsp90 molecular chaperone. Mol. Cell 42:96–105 [Google Scholar]
  66. Taipale M, Krykbaeva I, Koeva M, Kayatekin C, Westover KD. 66.  et al. 2012. Quantitative analysis of HSP90–client interactions reveals principles of substrate recognition. Cell 150:987–1001 [Google Scholar]
  67. Tan S, Davey CA. 67.  2011. Nucleosome structural studies. Curr. Opin. Struct. Biol. 21:128–36 [Google Scholar]
  68. Teilum K, Brath U, Lundström P, Akke M. 68.  2006. Biosynthetic 13C labeling of aromatic side chains in proteins for NMR relaxation measurements. J. Am. Chem. Soc. 128:2506–7 [Google Scholar]
  69. Tompa P, Fuxreiter M. 69.  2008. Fuzzy complexes: polymorphism and structural disorder in protein–protein interactions. Trends Biochem. Sci. 33:2–8 [Google Scholar]
  70. Tugarinov V, Choy WY, Orekhov VY, Kay LE. 70.  2005. Solution NMR–derived global fold of a monomeric 82-kDa enzyme. PNAS 102:622–27 [Google Scholar]
  71. Tugarinov V, Kanelis V, Kay LE. 71.  2006. Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nat. Protoc. 1:749–54 [Google Scholar]
  72. Tzeng SR, Kalodimos CG. 72.  2011. Protein dynamics and allostery: an NMR view. Curr. Opin. Struct. Biol. 21:62–67 [Google Scholar]
  73. Tzeng SR, Kalodimos CG. 73.  2012. Protein activity regulation by conformational entropy. Nature 488:236–40 [Google Scholar]
  74. Tzeng SR, Kalodimos CG. 74.  2013. Allosteric inhibition through suppression of transient conformational states. Nat. Chem. Biol. 9:462–65 [Google Scholar]
  75. Tzeng SR, Pai MT, Kalodimos CG. 75.  2012. NMR studies of large protein systems. Methods Mol. Biol. 831:133–40 [Google Scholar]
  76. Ullers RS, Ang D, Schwager F, Georgopoulos C, Genevaux P. 76.  2007. Trigger factor can antagonize both SecB and DnaK/DnaJ chaperone functions in Escherichia coli. PNAS 104:3101–6 [Google Scholar]
  77. Ullers RS, Luirink J, Harms N, Schwager F, Georgopoulos C, Genevaux P. 77.  2004. SecB is a bona fide generalized chaperone in Escherichia coli. PNAS 101:7583–88 [Google Scholar]
  78. Verba KA, Wang RY, Arakawa A, Liu Y, Shirouzu M. 78.  et al. 2016. Atomic structure of Hsp90–Cdc37–Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase. Science 352:1542–47 [Google Scholar]
  79. Von Eichborn J, Gunther S, Preissner R. 79.  2010. Structural features and evolution of protein–protein interactions. Genome Inform 22:1–10 [Google Scholar]
  80. von Heijne G. 80.  1985. Signal sequences: the limits of variation. J. Mol. Biol. 184:99–105 [Google Scholar]
  81. Whitty A. 81.  2008. Cooperativity and biological complexity. Nat. Chem. Biol. 4:435–39 [Google Scholar]
  82. Winkler J, Tyedmers J, Bukau B, Mogk A. 82.  2012. Chaperone networks in protein disaggregation and prion propagation. J. Struct. Biol. 179:152–60 [Google Scholar]
  83. Xu Z, Knafels JD, Yoshino K. 83.  2000. Crystal structure of the bacterial protein export chaperone secB. Nat. Struct. Biol. 7:1172–77 [Google Scholar]
  84. Zhuravleva A, Clerico EM, Gierasch LM. 84.  2012. An interdomain energetic tug-of-war creates the allosterically active state in Hsp70 molecular chaperones. Cell 151:1296–307 [Google Scholar]

Data & Media loading...

  • Article Type: Review Article
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error