Journal of Biomolecular NMR

, 51:21 | Cite as

Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis

  • Alexander I. Greenwood
  • Monique J. Rogals
  • Soumya De
  • Kun Ping Lu
  • Evgenii L. Kovrigin
  • Linda K. Nicholson


The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, \( k_{cat}^{cis} \) and apparent Michaelis constants, \( K_{M}^{App} \). By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific 13C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide 13C–1H constant time HSQC spectra to determine \( k_{cat}^{cis} \), \( k_{cat}^{trans} \), \( K_{D}^{cis} \), and \( K_{D}^{trans} \) for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.


Proyl isomerase Pin1 Lineshape analysis Isomerization Proline APP 



Financial support was provided by the US National Institutes of Health (R01-AG029385).

Supplementary material

10858_2011_9538_MOESM1_ESM.pdf (251 kb)
Supplementary material 1 (PDF 252 kb)


  1. Aliev AE, Courtier-Murias D (2007) Conformational analysis of L-prolines in water. J Phys Chem B 111:14034–14042CrossRefGoogle Scholar
  2. Anderson P (2005) Pin1: a proline isomerase that makes you wheeze? Nat Immunol 6:1211–1212CrossRefGoogle Scholar
  3. Ayed A, Mulder FA, Yi GS, Lu Y, Kay LE, Arrowsmith CH (2001) Latent and active p53 are identical in conformation. Nat Struct Biol 8:756–760CrossRefGoogle Scholar
  4. Bax AD, Davis DG (1985) Practical aspects of two-dimensional transverse NOE spectroscopy. J Magn Reson 63:207–213CrossRefGoogle Scholar
  5. Bosco DA, Eisenmesser EZ, Clarkson MW, Wolf-Watz M, Labeikovsky W, Millet O, Kern D (2010) Dissecting the microscopic steps of the cyclophilin A enzymatic cycle on the biological HIV-1 capsid substrate by NMR. J Mol Biol 403:723–738CrossRefGoogle Scholar
  6. Buratowski S (2003) The CTD code. Nat Struct Biol 10:679–680CrossRefGoogle Scholar
  7. Burbaum JJ, Raines RT, Albery WJ, Knowles JR (1989) Evolutionary optimization of the catalytic effectiveness of an enzyme. Biochemistry 28:9293–9305CrossRefGoogle Scholar
  8. Cavanagh J, Fairbrother WJ, Palmer AG, Rance M, Skelton NJ (2007) Protein NMR spectroscopy: principles and practice, 2nd edn. Academic Press, LondonGoogle Scholar
  9. Craven CJ, Whitehead B, Jones SK, Thulin E, Blackburn GM, Waltho JP (1996) Complexes formed between calmodulin and the antagonists J-8 and TFP in solution. Biochemistry 35:10287–10299CrossRefGoogle Scholar
  10. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  11. Dugave C, Demange L (2003) Cis-trans isomerization of organic molecules and biomolecules: implications and applications. Chem Rev 103:2475–2532CrossRefGoogle Scholar
  12. Eckerdt F, Yuan J, Saxena K, Martin B, Kappel S, Lindenau C, Kramer A, Naumann S, Daum S, Fischer G (2005) Polo-like kinase 1-mediated phosphorylation stabilizes Pin1 by inhibiting its ubiquitination in human cells. J Biol Chem 280:36575CrossRefGoogle Scholar
  13. Fanghanel J (2003) Enzymatic catalysis of the peptidyl-prolyl bond rotation: are transition state formation and enzyme dynamics directly linked? Angew Chem Int Ed Engl 42:490–492CrossRefGoogle Scholar
  14. Fischer G, Aumüller T (2003) Regulation of peptide bond cis/trans isomerization by enzyme catalysis and its implication in physiological processes. Rev Phys Biochem Pharmacol 148:105–150Google Scholar
  15. Fischer G, Heins J, Barth A (1983) The conformation around the peptide bond between the P1-and P2-positions is important for catalytic activity of some proline-specific proteases. Biochim Biophys Acta 742:452–462CrossRefGoogle Scholar
  16. Fischer G, Bang H, Berger E, Schellenberger A (1984) Conformational specificity of chymotrypsin toward proline-containing substrates. Biochim Biophys Acta 791:87–97CrossRefGoogle Scholar
  17. Fischer S, Michnick S, Karplus M (1993) A mechanism for rotamase catalysis by the FK506 binding protein (FKBP). Biochemistry 32:13830–13837CrossRefGoogle Scholar
  18. Garcia-Echeverria C, Kofron JL, Kuzmic P, Kishore V, Rich DH (1992) Continuous fluorimetric direct (uncoupled) assay for peptidyl prolyl cis-trans isomerases. J Am Chem Soc 114:2758–2759CrossRefGoogle Scholar
  19. Garcia-Echeverria C, Kofron JL, Kuzmic P, Rich DH (1993) A continuous spectrophotometric direct assay for peptidyl prolyl cis-trans isomerases. Biochem Biophys Res Commun 191:70–75CrossRefGoogle Scholar
  20. Goddard TD, Kneller DG (2008) Sparky 3. University of California, San FransciscoGoogle Scholar
  21. Grathwohl C, Wüthrich K (1981) NMR studies of the rates of proline cis-trans isomerization in oligopeptides. Biopolymers 20:2623–2633CrossRefGoogle Scholar
  22. Grishaev A, Tugarinov V, Kay LE, Trewhella J, Bax A (2008) Refined solution structure of the 82-kDa enzyme malate synthase G from joint NMR and synchrotron SAXS restraints. J Biomol NMR 40:95–106CrossRefGoogle Scholar
  23. Gunther UL, Schaffhausen B (2002) NMRKIN: simulating line shapes from two-dimensional spectra of proteins upon ligand binding. J Biomol NMR 22:201–209CrossRefGoogle Scholar
  24. Hansen DF, Vallurupalli P, Kay LE (2008a) An improved 15 N relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins. J Phys Chem B 112:5898–5904CrossRefGoogle Scholar
  25. Hansen DF, Vallurupalli P, Lundström P, Neudecker P, Kay LE (2008b) Probing chemical shifts of invisible states of proteins with relaxation dispersion NMR spectroscopy: how well can we do? J Am Chem Soc 130:2667–2675CrossRefGoogle Scholar
  26. Iijima K, Ando K, Takeda S, Satoh Y, Seki T, Itohara S, Greengard P, Kirino Y, Nairn AC, Suzuki T (2000) Neuron specific phosphorylation of Alzheimer’s amyloid precursor protein by cyclin dependent kinase 5. J Neurochem 75:1085–1091CrossRefGoogle Scholar
  27. Johnson PE, Creagh AL, Brun E, Joe K, Tomme P, Haynes CA, McIntosh LP (1998) Calcium binding by the N-terminal cellulose-binding domain from Cellulomonas fimi -1, 4-glucanase CenC. Biochemistry 37:12772–12781CrossRefGoogle Scholar
  28. Kern D, Kern G, Scherer G, Fischer G, Drakenberg T (1995) Kinetic analysis of cyclophilin-catalyzed prolyl cis/trans isomerization by dynamic NMR spectroscopy. Biochemistry 34:13594–13602CrossRefGoogle Scholar
  29. Kim SJ, Koh K, Lustig M, Boyd S, Gorinevsky D (2007) An interior-point method for large-scale l1-regularized least squares. IEEE J Sel Top Signal Process 1:606–617ADSCrossRefGoogle Scholar
  30. Kofron JL, Kuzmic P, Kishore V, Colon-Bonilla E, Rich DH (1991) Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay. Biochemistry 30:6127–6134CrossRefGoogle Scholar
  31. Korzhnev DM, Kay LE (2008) Probing invisible, low-populated states of protein molecules by relaxation dispersion NMR spectroscopy: an application to protein folding. Acc Chem Res 41:442–451CrossRefGoogle Scholar
  32. Kovrigin EL, Loria JP (2006) Enzyme dynamics along the reaction coordinate: critical role of a conserved residue. Biochemistry 45:2636–2647CrossRefGoogle Scholar
  33. Labeikovsky W, Eisenmesser EZ, Bosco DA, Kern D (2007) Structure and dynamics of pin1 during catalysis by NMR. J Mol Biol 367:1370–1381CrossRefGoogle Scholar
  34. Landrieu I, De Veylder L, Fruchart JS, Odaert B, Casteels P, Portetelle D, Van Montagu M, Inzé D, Lippens G (2000) The Arabidopsis thaliana PIN1At gene encodes a single-domain phosphorylation-dependent peptidyl prolylcis/trans isomerase. J Biol Chem 275:10577CrossRefGoogle Scholar
  35. Lee MS, Kao SC, Lemere CA, Xia W, Tseng HC, Zhou Y, Neve R, Ahlijanian MK, Tsai LH (2003) APP processing is regulated by cytoplasmic phosphorylation. J Cell Biol 163:83–95CrossRefGoogle Scholar
  36. Li P, Martins IR, Amarasinghe GK, Rosen MK (2008) Internal dynamics control activation and activity of the autoinhibited Vav DH domain. Nat Struct Mol Biol 15:613–618CrossRefGoogle Scholar
  37. Lin LN, Brandts JF (1985) Isomer-specific proteolysis of model substrates: influence that the location of the proline residue exerts on cis trans specificity. Biochemistry 24:6533–6538CrossRefGoogle Scholar
  38. Lu KP, Zhou XZ (2007) The prolyl isomerase PIN1: a pivotal new twist in phosphorylation signalling and disease. Nat Rev Mol Cell Biol 8:904–916CrossRefGoogle Scholar
  39. Lu KP, Hanes SD, Hunter T (1996) A human peptidyl-prolyl isomerase essential for regulation of mitosis. Nature 380:544–547ADSCrossRefGoogle Scholar
  40. Lu PJ, Zhou XZ, Liou YC, Noel JP, Lu KP (2002) Critical role of WW domain phosphorylation in regulating phosphoserine binding activity and Pin1 function. J Biol Chem 277:2381CrossRefGoogle Scholar
  41. Lu KP, Suizu F, Zhou XZ, Finn G, Lam P, Wulf G (2006) Targeting carcinogenesis: a role for the prolyl isomerase Pin1? Mol Carcinog 45:397–402CrossRefGoogle Scholar
  42. Lu KP, Finn G, Lee TH, Nicholson LK (2007) Prolyl cis-trans isomerization as a molecular timer. Nat Chem Biol 3:619–629CrossRefGoogle Scholar
  43. Massi F, Johnson E, Wang C, Rance M, Palmer AG III (2004) NMR R1 rho rotating-frame relaxation with weak radio frequency fields. J Am Chem Soc 126:2247–2256CrossRefGoogle Scholar
  44. McConnell HM (1958) Reaction rates by nuclear magnetic resonance. J Chem Phys 28:430ADSCrossRefGoogle Scholar
  45. Meinhart A, Cramer P (2004) Recognition of RNA polymerase II carboxy-terminal domain by 3’-RNA-processing factors. Nature 430:223–226ADSCrossRefGoogle Scholar
  46. Meraz-Rios MA, Lira‐De León KI, Campos-Pena V, De Anda-Hernandez MA, Mena-Lopez R (2010) Tau oligomers and aggregates in Alzheimer’s disease. J Neurochem 112:1353–1367CrossRefGoogle Scholar
  47. Mittermaier AK, Kay LE (2009) Observing biological dynamics at atomic resolution using NMR. Trends Biochem Sci 34:601–611CrossRefGoogle Scholar
  48. Mulder FAA, Spronk CAEM, Slijper M, Kaptein R, Boelens R (1996) Improved HSQC experiments for the observation of exchange broadened signals. J Biomol NMR 8:223–228CrossRefGoogle Scholar
  49. Mulder FA, Schipper D, Bott R, Boelens R (1999) Altered flexibility in the substrate-binding site of related native and engineered high-alkaline Bacillus subtilisins. J Mol Biol 292:111–123CrossRefGoogle Scholar
  50. Park ST, Aldape RA, Futer O, DeCenzo MT, Livingston DJ (1992) PPIase catalysis by human FK506-binding protein proceeds through a conformational twist mechanism. J Biol Chem 267:3316–3324Google Scholar
  51. Pastorino L, Sun A, Lu PJ, Zhou XZ, Balastik M, Finn G, Wulf G, Lim J, Li SH, Li X et al (2006) The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-beta production. Nature 440:528–534ADSCrossRefGoogle Scholar
  52. Peng JW, Wilson BD, Namanja AT (2009) Mapping the dynamics of ligand reorganization via 13 CH 3 and 13 CH 2 relaxation dispersion at natural abundance. J Biomol NMR 45:171–183CrossRefGoogle Scholar
  53. Ramelot TA, Nicholson LK (2001) Phosphorylation-induced structural changes in the amyloid precursor protein cytoplasmic tail detected by NMR. J Mol Biol 307:871–884CrossRefGoogle Scholar
  54. Ramelot TA, Gentile LN, Nicholson LK (2000) Transient structure of the amyloid precursor protein cytoplasmic tail indicates preordering of structure for binding to cytosolic factors. Biochemistry 39:2714–2725CrossRefGoogle Scholar
  55. Ranganathan R, Lu KP, Hunter T, Noel JP (1997) Structural and functional analysis of the mitotic rotamase Pin1 suggests substrate recognition is phosphorylation dependent. Cell 89:875–886CrossRefGoogle Scholar
  56. Reimer U, Scherer G, Drewello M, Kruber S, Schutkowski M, Fischer G (1998) Side-chain effects on peptidyl-prolyl cis/trans isomerisation. J Mol Biol 279:449–460CrossRefGoogle Scholar
  57. Religa TL, Sprangers R, Kay LE (2010) Dynamic regulation of archaeal proteasome gate opening as studied by TROSY NMR. Science 328:98ADSCrossRefGoogle Scholar
  58. Rippmann JF, Hobbie S, Daiber C, Guilliard B, Bauer M, Birk J, Nar H, Garin-Chesa P, Rettig WJ, Schnapp A (2000) Phosphorylation-dependent proline isomerization catalyzed by Pin1 is essential for tumor cell survival and entry into mitosis. Cell Growth Differ Mol Biol J Am Assoc Cancer Res 11:409Google Scholar
  59. Sarkar P, Saleh T, Tzeng SR, Birge RB, Kalodimos CG (2011) Structural basis for regulation of the Crk signaling protein by a proline switch. Nat Chem Biol 7:51–57CrossRefGoogle Scholar
  60. Schutkowski M, Bernhardt A, Zhou XZ, Shen M, Reimer U, Rahfeld JU, Lu KP, Fischer G (1998) Role of phosphorylation in determining the backbone dynamics of the serine/threonine-proline motif and Pin1 substrate recognition. Biochemistry 37:5566–5575CrossRefGoogle Scholar
  61. Skinner GM, Baumann CG, Quinn DM, Molloy JE, Hoggett JG (2004) Promoter binding, initiation, and elongation by bacteriophage T7 RNA polymerase. A single-molecule view of the transcription cycle. J Biol Chem 279:3239–3244CrossRefGoogle Scholar
  62. Sprangers R, Kay LE (2007) Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 445:618–622CrossRefGoogle Scholar
  63. Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447:1021–1025ADSCrossRefGoogle Scholar
  64. Vallurupalli P, Hansen DF, Kay LE (2008) Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy. Proc Natl Acad Sci 105:11766ADSCrossRefGoogle Scholar
  65. Verdecia MA, Bowman ME, Lu KP, Hunter T, Noel JP (2000) Structural basis for phosphoserine-proline recognition by group IV WW domains. Nat Struct Biol 7:639–643CrossRefGoogle Scholar
  66. Vuister GW, Bax A (1992) Resolution enhancement and spectral editing of uniformy 13C-enriched proteins by homonuclear braodband 13C decoupling. J Magn Reson 98:428–435CrossRefGoogle Scholar
  67. Wang C, Palmer AG III (2003) Solution NMR methods for quantitative identification of chemical exchange in 15 N labeled proteins. Magn Reson Chem 41:866–876CrossRefGoogle Scholar
  68. Werner-Allen JW, Lee CJ, Liu P, Nicely NI, Wang S, Greenleaf AL, Zhou P (2011) cis-Proline-mediated Ser(P)5 dephosphorylation by the RNA polymerase II C-terminal domain phosphatase Ssu72. J Biol Chem 286:5717–5726CrossRefGoogle Scholar
  69. Xiang K, Nagaike T, Xiang S, Kilic T, Beh MM, Manley JL, Tong L (2010) Crystal structure of the human symplekin-Ssu72-CTD phosphopeptide complex. Nature 467:729–733ADSCrossRefGoogle Scholar
  70. Zhou XZ, Kops O, Werner A, Lu PJ, Shen M, Stoller G, Kullertz G, Stark M, Fischer G, Lu KP (2000) Pin1-dependent prolyl isomerization regulates dephosphorylation of Cdc25C and tau proteins. Mol Cell 6:873–883CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Alexander I. Greenwood
    • 1
  • Monique J. Rogals
    • 1
  • Soumya De
    • 1
  • Kun Ping Lu
    • 2
  • Evgenii L. Kovrigin
    • 3
    • 4
  • Linda K. Nicholson
    • 1
  1. 1.Department of Molecular Biology and GeneticsCornell UniversityIthacaUSA
  2. 2.Cancer Biology Program and Biology of Aging Program, Beth Israel Deaconess Medical Center, Harvard Medical SchoolBostonUSA
  3. 3.Department of BiochemistryMedical College of WisconsinMilwaukeeUSA
  4. 4.Chemistry DepartmentMarquette UniversityMilwaukeeUSA

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