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Disorder for Dummies: Functional Mutagenesis of Transient Helical Segments in Disordered Proteins

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Intrinsically Disordered Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2141))

Abstract

Most cytosolic eukaryotic proteins contain a mixture of ordered and disordered regions. Disordered regions facilitate cell signaling by concentrating sites for posttranslational modifications and protein–protein interactions into arrays of short linear motifs that can be reorganized by RNA splicing. The evolution of disordered regions looks different from their ordered counterparts. In some cases, selection is focused on maintaining protein binding interfaces and PTM sites, but sequence heterogeneity is common. In other cases, simple properties like charge, length, or end-to-end distance are maintained. Many disordered protein binding sites contain some transient secondary structure that may resemble the structure of the bound state. α-Helical secondary structure is common and a wide range of fractional helicity is observed in different disordered regions. Here we provide a simple protocol to identify transient helical segments and design mutants that can change their structure and function.

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References

  1. Romero P, Obradovic Z, Kissinger CR et al (1998) Thousands of proteins likely to have long disordered regions. Pac Symp Biocomput 3:437–448

    Google Scholar 

  2. Dunker AK, Obradovic Z, Romero P et al (2000) Intrinsic protein disorder in complete genomes. Genome Inform Ser Workshop Genome Inform 11:161–171

    CAS  PubMed  Google Scholar 

  3. Dunker AK, Lawson JD, Brown CJ et al (2001) Intrinsically disordered protein. J Mol Graph Model 19(1):26–59

    Article  CAS  PubMed  Google Scholar 

  4. Kriwacki RW, Hengst L, Tennant L et al (1996) Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity. Proc Natl Acad Sci U S A 93(21):11504–11509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lefevre JF, Dayie KT, Peng JW et al (1996) Internal mobility in the partially folded DNA binding and dimerization domains of GAL4: NMR analysis of the N-H spectral density functions. Biochemistry 35(8):2674–2686

    Article  CAS  PubMed  Google Scholar 

  6. Daughdrill GW, Chadsey MS, Karlinsey JE et al (1997) The C-terminal half of the anti-sigma factor, FlgM, becomes structured when bound to its target, sigma 28. Nat Struct Biol 4(4):285–291

    Article  CAS  PubMed  Google Scholar 

  7. Radhakrishnan I, Perez-Alvarado GC, Parker D et al (1997) Solution structure of the KIX domain of CBP bound to the transactivation domain of CREB: a model for activator:coactivator interactions. Cell 91(6):741–752

    Article  CAS  PubMed  Google Scholar 

  8. Donne DG, Viles JH, Groth D et al (1997) Structure of the recombinant full-length hamster prion protein PrP(29-231): the N terminus is highly flexible. Proc Natl Acad Sci U S A 94(25):13452–13457

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wright PE, Dyson HJ (1999) Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. J Mol Biol 293(2):321–331

    Article  CAS  PubMed  Google Scholar 

  10. van der Lee R, Buljan M, Lang B et al (2014) Classification of intrinsically disordered regions and proteins. Chem Rev 114(13):6589–6631. https://doi.org/10.1021/cr400525m

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Daughdrill GW, Pielak GJ, Uversky VN et al (2005) Natively disordered proteins. In: Buchner J, Kiefhaber T (eds) Protein folding handbook, vol 3. WILEY-VCH, Darmstadt, pp 275–357

    Chapter  Google Scholar 

  12. Vise PD, Baral B, Latos AJ et al (2005) NMR chemical shift and relaxation measurements provide evidence for the coupled folding and binding of the p53 transactivation domain. Nucleic Acids Res 33(7):2061–2077

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Daughdrill GW, Narayanaswami P, Gilmore SH et al (2007) Dynamic behavior of an intrinsically unstructured linker domain is conserved in the face of negligible amino acid sequence conservation. J Mol Evol 65(3):277–288

    Article  CAS  PubMed  Google Scholar 

  14. Gely S, Lowry DF, Bernard C et al (2010) Solution structure of the C-terminal X domain of the measles virus phosphoprotein and interaction with the intrinsically disordered C-terminal domain of the nucleoprotein. J Mol Recognit 23(5):435–447. https://doi.org/10.1002/jmr.1010

    Article  CAS  PubMed  Google Scholar 

  15. Dyson HJ, Wright PE (2001) Nuclear magnetic resonance methods for elucidation of structure and dynamics in disordered states. Methods Enzymol 339:258–270

    Article  CAS  PubMed  Google Scholar 

  16. Dyson HJ, Wright PE (2002) Coupling of folding and binding for unstructured proteins. Curr Opin Struct Biol 12(1):54–60

    Article  CAS  PubMed  Google Scholar 

  17. Cheng Y, Oldfield CJ, Meng J et al (2007) Mining alpha-helix-forming molecular recognition features with cross species sequence alignments. Biochemistry 46(47):13468–13477. https://doi.org/10.1021/bi7012273

    Article  CAS  PubMed  Google Scholar 

  18. Mohan A, Oldfield CJ, Radivojac P et al (2006) Analysis of molecular recognition features (MoRFs). J Mol Biol 362(5):1043–1059. https://doi.org/10.1016/j.jmb.2006.07.087

    Article  CAS  PubMed  Google Scholar 

  19. Oldfield CJ, Cheng Y, Cortese MS et al (2005) Coupled folding and binding with alpha-helix-forming molecular recognition elements. Biochemistry 44(37):12454–12470

    Article  CAS  PubMed  Google Scholar 

  20. Fuxreiter M, Simon I, Friedrich P et al (2004) Preformed structural elements feature in partner recognition by intrinsically unstructured proteins. J Mol Biol 338(5):1015–1026

    Article  CAS  PubMed  Google Scholar 

  21. Dyson HJ (2013) Coupled folding and binding. In: Roberts GCK (ed) Encyclopedia of biophysics. Springer Berlin Heidelberg, Berlin, Heidelberg, pp 381–385. https://doi.org/10.1007/978-3-642-16712-6_174

    Chapter  Google Scholar 

  22. Gianni S, Dogan J, Jemth P (2016) Coupled binding and folding of intrinsically disordered proteins: what can we learn from kinetics? Curr Opin Struct Biol 36:18–24. https://doi.org/10.1016/j.sbi.2015.11.012

    Article  CAS  PubMed  Google Scholar 

  23. Spolar RS, Record MT Jr (1994) Coupling of local folding to site-specific binding of proteins to DNA. Science 263(5148):777–784

    Article  CAS  PubMed  Google Scholar 

  24. Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447(7147):1021–1025

    Article  CAS  PubMed  Google Scholar 

  25. Munoz V, Serrano L (1994) Elucidating the folding problem of helical peptides using empirical parameters. Nat Struct Biol 1(6):399–409

    Article  CAS  PubMed  Google Scholar 

  26. Lacroix E, Viguera AR, Serrano L (1998) Elucidating the folding problem of alpha-helices: local motifs, long-range electrostatics, ionic-strength dependence and prediction of NMR parameters. J Mol Biol 284(1):173–191. https://doi.org/10.1006/jmbi.1998.2145

    Article  CAS  PubMed  Google Scholar 

  27. Sowemimo OT, Knox-Brown P, Borcherds W et al (2019) Conserved Glycines control disorder and function in the cold-regulated protein, COR15A. Biomol Ther 9(3). https://doi.org/10.3390/biom9030084

  28. Kennedy JA, Daughdrill GW, Schmidt KH (2013) A transient alpha-helical molecular recognition element in the disordered N-terminus of the Sgs1 helicase is critical for chromosome stability and binding of Top3/Rmi1. Nucleic Acids Res 41(22):10215–10227. https://doi.org/10.1093/nar/gkt817

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Borcherds W, Theillet FX, Katzer A et al (2014) Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells. Nat Chem Biol 10(12):1000–1002. https://doi.org/10.1038/nchembio.1668

    Article  CAS  PubMed  Google Scholar 

  30. Poosapati A, Gregory E, Borcherds WM et al (2018) Uncoupling the folding and binding of an intrinsically disordered protein. J Mol Biol 430(16):2389–2402. https://doi.org/10.1016/j.jmb.2018.05.045

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Chen L, Borcherds W, Wu S et al (2015) Autoinhibition of MDMX by intramolecular p53 mimicry. Proc Natl Acad Sci U S A 112(15):4624–4629. https://doi.org/10.1073/pnas.1420833112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Brown CJ, Takayama S, Campen AM et al (2002) Evolutionary rate heterogeneity in proteins with long disordered regions. J Mol Evol 55(1):104–110

    Article  CAS  PubMed  Google Scholar 

  33. Radivojac P, Obradovic Z, Brown CJ et al (2002) Improving sequence alignments for intrinsically disordered proteins. In: Pac Symp Biocomput, pp 589–600

    Google Scholar 

  34. Brown CJ, Johnson AK, Daughdrill GW (2010) Comparing models of evolution for ordered and disordered proteins. Mol Biol Evol 27(3):609–621. https://doi.org/10.1093/molbev/msp277

    Article  CAS  PubMed  Google Scholar 

  35. Ahrens JB, Rahaman J, Siltberg-Liberles J (2018) Large-scale analyses of site-specific evolutionary rates across eukaryote proteomes reveal confounding interactions between intrinsic disorder, secondary structure, and functional domains. Genes (Basel) 9(11):E553. https://doi.org/10.3390/genes9110553

    Article  CAS  Google Scholar 

  36. Gunasekaran K, Tsai CJ, Nussinov R (2004) Analysis of ordered and disordered protein complexes reveals structural features discriminating between stable and unstable monomers. J Mol Biol 341(5):1327–1341. https://doi.org/10.1016/j.jmb.2004.07.002

    Article  CAS  PubMed  Google Scholar 

  37. Borcherds W, Kashtanov S, Wu H et al (2013) Structural divergence is more extensive than sequence divergence for a family of intrinsically disordered proteins. Proteins 81(10):1686–1698. https://doi.org/10.1002/prot.24303

    Article  CAS  PubMed  Google Scholar 

  38. Higgins DG, Thompson JD, Gibson TJ (1996) Using CLUSTAL for multiple sequence alignments. Methods Enzymol 266:383–402

    Article  CAS  PubMed  Google Scholar 

  39. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673–4680. https://doi.org/10.1093/nar/22.22.4673

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Dosztanyi Z, Csizmok V, Tompa P et al (2005) The pairwise energy content estimated from amino acid composition discriminates between folded and intrinsically unstructured proteins. J Mol Biol 347(4):827–839

    Article  CAS  PubMed  Google Scholar 

  41. Meszaros B, Erdos G, Dosztanyi Z (2018) IUPred2A: context-dependent prediction of protein disorder as a function of redox state and protein binding. Nucleic Acids Res 46(W1):W329–W337. https://doi.org/10.1093/nar/gky384

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Deng X, Eickholt J, Cheng J (2012) A comprehensive overview of computational protein disorder prediction methods. Mol BioSyst 8(1):114–121. https://doi.org/10.1039/c1mb05207a

    Article  CAS  PubMed  Google Scholar 

  43. Lieutaud P, Ferron F, Uversky AV et al (2016) How disordered is my protein and what is its disorder for? A guide through the "dark side" of the protein universe. Intrinsically Disord Proteins 4(1):e1259708. https://doi.org/10.1080/21690707.2016.1259708

    Article  PubMed  PubMed Central  Google Scholar 

  44. Dawson R, Muller L, Dehner A et al (2003) The N-terminal domain of p53 is natively unfolded. J Mol Biol 332(5):1131–1141

    Article  CAS  PubMed  Google Scholar 

  45. Lee H, Mok KH, Muhandiram R et al (2000) Local structural elements in the mostly unstructured transcriptional activation domain of human p53. J Biol Chem 275(38):29426–29432

    Article  CAS  PubMed  Google Scholar 

  46. Laptenko O, Tong DR, Manfredi J et al (2016) The tail that wags the dog: how the disordered C-terminal domain controls the transcriptional activities of the p53 tumor-suppressor protein. Trends Biochem Sci 41(12):1022–1034. https://doi.org/10.1016/j.tibs.2016.08.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Ayed A, Mulder FA, Yi GS et al (2001) Latent and active p53 are identical in conformation. Nat Struct Biol 8(9):756–760. https://doi.org/10.1038/nsb0901-756

    Article  CAS  PubMed  Google Scholar 

  48. Weinberg RL, Freund SM, Veprintsev DB et al (2004) Regulation of DNA binding of p53 by its C-terminal domain. J Mol Biol 342(3):801–811. https://doi.org/10.1016/j.jmb.2004.07.042

    Article  CAS  PubMed  Google Scholar 

  49. Finch RA, Donoviel DB, Potter D et al (2002) Mdmx is a negative regulator of p53 activity in vivo. Cancer Res 62(11):3221–3225

    CAS  PubMed  Google Scholar 

  50. Migliorini D, Lazzerini Denchi E, Danovi D et al (2002) Mdm4 (Mdmx) regulates p53-induced growth arrest and neuronal cell death during early embryonic mouse development. Mol Cell Biol 22(15):5527–5538

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Popowicz GM, Czarna A, Holak TA (2008) Structure of the human Mdmx protein bound to the p53 tumor suppressor transactivation domain. Cell Cycle 7(15):2441–2443. https://doi.org/10.4161/cc.6365

    Article  CAS  PubMed  Google Scholar 

  52. Borcherds W, Becker A, Chen L et al (2017) Optimal affinity enhancement by a conserved flexible linker controls p53 mimicry in MdmX. Biophys J 112(10):2038–2042. https://doi.org/10.1016/j.bpj.2017.04.017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kennedy JA, Syed S, Schmidt KH (2015) Structural motifs critical for in vivo function and stability of the RecQ-mediated genome instability protein Rmi1. PLoS One 10(12):e0145466. https://doi.org/10.1371/journal.pone.0145466

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Harmon TS, Crabtree MD, Shammas SL et al (2016) GADIS: algorithm for designing sequences to achieve target secondary structure profiles of intrinsically disordered proteins. Protein Eng Des Sel 29(9):339–346. https://doi.org/10.1093/protein/gzw034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Lee SH, Kim DH, Lee SH et al (2012) Understanding pre-structured motifs (PreSMos) in intrinsically unfolded proteins. Curr Protein Pept Sci 13(1):34–54

    Article  CAS  PubMed  Google Scholar 

  56. Borcherds WM, Daughdrill GW (2018) Using NMR chemical shifts to determine residue-specific secondary structure populations for intrinsically disordered proteins. Methods Enzymol 611:101–136. https://doi.org/10.1016/bs.mie.2018.09.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Cho Y, Gorina S, Jeffrey PD et al (1994) Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 265(5170):346–355

    Article  CAS  PubMed  Google Scholar 

  58. Clore GM, Omichinski JG, Sakaguchi K et al (1994) High-resolution structure of the oligomerization domain of p53 by multidimensional NMR. Science 265(5170):386–391

    Article  CAS  PubMed  Google Scholar 

  59. Lee W, Harvey TS, Yin Y et al (1994) Solution structure of the tetrameric minimum transforming domain of p53. Nat Struct Biol 1(12):877–890

    Article  CAS  PubMed  Google Scholar 

  60. Fang S, Jensen JP, Ludwig RL et al (2000) Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. J Biol Chem 275(12):8945–8951. https://doi.org/10.1074/jbc.275.12.8945

    Article  CAS  PubMed  Google Scholar 

  61. Camilloni C, De Simone A, Vranken WF et al (2012) Determination of secondary structure populations in disordered states of proteins using nuclear magnetic resonance chemical shifts. Biochemistry 51(11):2224–2231. https://doi.org/10.1021/bi3001825

    Article  CAS  PubMed  Google Scholar 

  62. Lee C, Kalmar L, Xue B et al (2014) Contribution of proline to the pre-structuring tendency of transient helical secondary structure elements in intrinsically disordered proteins. Biochim Biophys Acta 1840(3):993–1003. https://doi.org/10.1016/j.bbagen.2013.10.042

    Article  CAS  PubMed  Google Scholar 

  63. Crabtree MD, Borcherds W, Poosapati A et al (2017) Conserved helix-flanking Prolines modulate intrinsically disordered protein:target affinity by altering the lifetime of the bound complex. Biochemistry 56(18):2379–2384. https://doi.org/10.1021/acs.biochem.7b00179

    Article  CAS  PubMed  Google Scholar 

  64. Watt PM, Louis EJ, Borts RH et al (1995) Sgs1: a eukaryotic homolog of E. coli RecQ that interacts with topoisomerase II in vivo and is required for faithful chromosome segregation. Cell 81(2):253–260

    Article  CAS  PubMed  Google Scholar 

  65. Munoz V, Serrano L (1995) Elucidating the folding problem of helical peptides using empirical parameters. III. Temperature and pH dependence. J Mol Biol 245(3):297–308. https://doi.org/10.1006/jmbi.1994.0024

    Article  CAS  PubMed  Google Scholar 

  66. Wishart DS, Sykes BD, Richards FM (1991) Relationship between nuclear magnetic resonance chemical shift and protein secondary structure. J Mol Biol 222(2):311–333

    Article  CAS  PubMed  Google Scholar 

  67. Wishart DS, Sykes BD (1994) The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J Biomol NMR 4(2):171–180

    Article  CAS  PubMed  Google Scholar 

  68. Wishart DS, Bigam CG, Holm A et al (1995) H-1, C-13 and N-15 random coil Nmr chemical-shifts of the common amino-acids .1. Investigations of nearest-neighbor effects (Vol 5, Pg 67, 1995). J Biomol NMR 5(3):332–332

    Article  CAS  PubMed  Google Scholar 

  69. Nielsen JT, Mulder FAA (2018) POTENCI: prediction of temperature, neighbor and pH-corrected chemical shifts for intrinsically disordered proteins. J Biomol NMR 70(3):141–165. https://doi.org/10.1007/s10858-018-0166-5

    Article  CAS  PubMed  Google Scholar 

  70. Tamiola K, Acar B, Mulder FA (2010) Sequence-specific random coil chemical shifts of intrinsically disordered proteins. J Am Chem Soc 132(51):18000–18003. https://doi.org/10.1021/ja105656t

    Article  CAS  PubMed  Google Scholar 

  71. Kjaergaard M, Poulsen FM (2011) Sequence correction of random coil chemical shifts: correlation between neighbor correction factors and changes in the Ramachandran distribution. J Biomol NMR 50(2):157–165. https://doi.org/10.1007/s10858-011-9508-2

    Article  CAS  PubMed  Google Scholar 

  72. Zhang HY, Neal S, Wishart DS (2003) RefDB: a database of uniformly referenced protein chemical shifts. J Biomol NMR 25(3):173–195. https://doi.org/10.1023/A:1022836027055

    Article  CAS  PubMed  Google Scholar 

  73. De Simone A, Cavalli A, Hsu STD et al (2009) Accurate random coil chemical shifts from an analysis of loop regions in native states of proteins. J Am Chem Soc 131(45):16332. https://doi.org/10.1021/ja904937a

    Article  CAS  PubMed  Google Scholar 

  74. Schwarzinger S, Kroon GJA, Foss TR et al (2000) Random coil chemical shifts in acidic 8 M urea: implementation of random coil shift data in NMRView. J Biomol NMR 18(1):43–48. https://doi.org/10.1023/A:1008386816521

    Article  CAS  PubMed  Google Scholar 

  75. Dyson HJ, Wright PE (2002) Insights into the structure and dynamics of unfolded proteins from nuclear magnetic resonance. In: Unfolded proteins, vol 62. Advances in Protein Chemistry. Academic Press Inc, San Diego, pp 311–340

    Chapter  Google Scholar 

  76. Wishart DS (2011) Interpreting protein chemical shift data. Prog Nucl Magn Reson Spectrosc 58(1–2):62–87. https://doi.org/10.1016/j.pnmrs.2010.07.004

    Article  CAS  PubMed  Google Scholar 

  77. Neal S, Nip AM, Zhang HY et al (2003) Rapid and accurate calculation of protein H-1, C-13 and N-15 chemical shifts. J Biomol NMR 26(3):215–240. https://doi.org/10.1023/A:1023812930288

    Article  CAS  PubMed  Google Scholar 

  78. Wishart DS, Nip AM (1998) Protein chemical shift analysis: a practical guide. Biochem Cell Biol 76(2–3):153–163. https://doi.org/10.1139/bcb-76-2-3-153

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

G.W.D. is supported by the National Institutes of Health (CA14124406 and GM115556).

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Authors and Affiliations

  1. Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL, USA

    Gary W. Daughdrill

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  1. Gary W. Daughdrill
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Correspondence to Gary W. Daughdrill .

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Editors and Affiliations

  1. Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, København N, Denmark

    Birthe B. Kragelund

  2. Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark

    Karen Skriver

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Daughdrill, G.W. (2020). Disorder for Dummies: Functional Mutagenesis of Transient Helical Segments in Disordered Proteins. In: Kragelund, B.B., Skriver, K. (eds) Intrinsically Disordered Proteins. Methods in Molecular Biology, vol 2141. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0524-0_1

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  • DOI: https://doi.org/10.1007/978-1-0716-0524-0_1

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