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Intrinsically Disordered Proteins: from lack of structure to pleiotropy of functions

Intrinsically Disordered Proteins: from lack of structure to pleiotropy of functions. Lilia Iakoucheva University of California, San Diego. OUTLINE. Characterization and properties of IDPs Functional repertoire of IDPs Post-translational modifications and disorder

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Intrinsically Disordered Proteins: from lack of structure to pleiotropy of functions

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  1. Intrinsically Disordered Proteins: from lack of structure to pleiotropy of functions Lilia Iakoucheva University of California, San Diego

  2. OUTLINE • Characterization and properties of IDPs • Functional repertoire of IDPs • Post-translational modifications anddisorder • Importance for molecular recognition • Disorder and diseases

  3. Protein Structure-Function Paradigm Amino Acid Sequence 3D Structure Function Historical perspective 1894 - Emil Fischer’s “lock-and-key” hypothesis: 1950 – Fred Karush “Configurational adaptability” 1958 – Daniel Koshland “Induced fit” theory

  4. Disorder examples Some proteins/regions could function without being folded…= disordered First examples of disorder: • Tail of histone H5 (Aviles et al, Eur. J. Biochem. 1978) … and later tails of other histones • 95-residue long disordered segment of calcineurin • (Kissinger et al, Nature, 1995) • Cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 • (Kriwacki et al, PNAS, 1996) Etc…

  5. Order Disorder Function Amino Acid Sequence Amino Acid Sequence 3D Structure Function Re-assessing structure-function paradigm

  6. What is disorder? Protein regions (or entire proteins) lacking stable II and III structure and existing in the ensemble of conformations with dynamically changing Ramachandran angles Disorderis experimentally detected by • X-ray crystallography • NMR spectroscopy • circular dichroism (CD) • limited proteolysis (LP) • hydrodynamic methods Bracken et al, CurrOpinStruct Biol. 2004, 570; Receveur-Bréchot et al, Proteins, 2006, 24

  7. DISORDER “I don’t know about hair care, Rapunzel, but I’m thinking a good cream rinse plus PROTEIN conditioner might just solve both our problems.”

  8. Properties of IDRs and IDPs Compositional bias Charge-hydropathy plot Order- promoting Disorder- promoting ↓Aromatic, hydrophobic ↑Charged, hydrophilic ↑ Net charge ↓ Hydrophobicity ↓ Net charge ↑ Hydrophobicity Dunker et al, 2001, JMGM; Radivojac et al, 2007, Biophys J Uversky et al, 2000, Proteins 41:415-427

  9. Disorder prediction AA sequence codes for protein structure… Does AA sequence code for the lack of structure? Keith Dunker group – first Predictor Of Natural Disordered Regions PONDR • amino acid composition • sequence complexity • net charge • hydrophobicity • flexibility • …and other features

  10. http://www.disprot.org/ predictors.php

  11. PONDRing XPA XPA-MBD Structure of the full-length XPA ??? Ikegami et al,1998, Nat.Struct.Biol.

  12. PONDR in action Iakoucheva et al, Prot Science 2001

  13. P-site DDB2 Functional importance DNA BD TFIIH RPA RPA ERCC1 NLS NLS • Protein-protein interaction sites are mapped to disordered XPA termini • XPA’s phosphorylation site is located in its disordered C-terminus • Putative XPA nuclear localization signals (NLS) are located in disordered regions

  14. Disorder and Functions • Dunker et al, 2002, Biochemistry

  15. Advantages of being disordered • Low-affinity/high-specificity binding • Broad binding diversity • Ability to form large interaction surfaces • Greater capture radius (“fly-casting” mechanism) • Facilitate alternative splicing • Facilitate post-translational modifications

  16. Gsponer et al, 2008, Science. 322(5906):1365-8 Phos-sites prefer IDRs DisPhos http://core.ist.temple.edu/pred/ More kinases that target IDPs! More kinase targets are IDPs!

  17. Ubiquitination and disorder • IDPs are susceptible to proteasomal degradation • Unstructured initiation site is required for degradation (Prakash et al, 2004, Nat Struct Mol Biol.) • PEST motifs are disordered (Singh et al, 2006, Proteins) • Low coverage of known Ub sites by PDB • Examples of Ub sites in IDRs (p53, c-myc, cyclin B, securin, p21, p27, p57, α-synuclein, IκBα etc, various authors)

  18. Ubligases β-catenin peptide: 15 out of 26 aa are disordered Wu et al, 2003, Molecular Cell, Vol. 11, 1445–1456 ~60A° gap p27 peptide: 14 out of 24 aa are disordered Hao et al, 2005, Molecular Cell, Vol. 20, 9-19

  19. Ub sites properties Identified 145 new Ub sites with MudPit, mass-spec SILAC and mutant (grr1Δ and cdc34tm) yeast strains to target short-lived proteins Ub sites: Negative charge D and E K and hydrophobics Disorder Predicted B-factors

  20. UbPred http://www.UbPred.org Radivojac et al, Proteins, 2010 Radivojac et al, Proteins, 2010

  21. Dynamic disorder of Sic1 bound to Cdc4 Structural Model of the Dynamic pSic1-Cdc4 Complex Sic1 contains 9 phosphorylation sites, which interact with Cdc4 in a dynamic equilibrium Directly interacting residues are transiently ordered, whereas the rest of Sic1 remains disordered even in the complex The disorder of Sic1 helps to bridge the 64A gap between E2 (Cdc34) and the Sic1 bound to Cdc4 for ubiquitin transfer Mittag et al, Structure, 2010

  22. Disorder and Functions

  23. Molecular recognition Disordered regions are commonly used for binding to multiple partners… C-terminus of p53 NCBD domain of CBP/p300 Oldfield et al, 2008, BMC Genomics. 9 Suppl 1:S1 Wright and Dyson, 2009, Curr Opin Struct Biol.

  24. Mechanisms of binding for IDPs How do disordered proteins bind to their targets? Induced folding (binding, … then folding) Conformational selection (folding, … then binding) Coupled/synergistic (folding and binding, … or even binding without folding) (CFTR R and NBD1 domains, Baker et al, 2007, Nat Struct Mol Biol, 14:738)

  25. Hubs and disorder Are disordered proteins network hubs? Cytoskeletal hubs subnetwork from the S.cerevisiaeinteractome Yeast PPI Haynes et al, 2006, PLoS CB

  26. Ordered hubs – disordered partners 14-3-3 proteins – signal transduction, apoptosis, cell cycle, cancer >200 binding (mostly phosphorylated) targets Three different predictors indicate that 14-3-3 TARGETSare highly disordered (Bustos and Iglesias, 2006, Proteins, 63:35–42) Peptides bind to essentially the same region of 14-3-3 Differences in 14-3-3 side chains conformations (e.g. induced fit mechanism) Peptides are highly hydrated in the bound state (e.g. likely disordered in the unbound state) Oldfield et al, BMC Genomics, 2008, 9(Suppl 1):S1

  27. Disorder and disease Individual examples of IDPs/IDRs involved in human diseases: p53 (cancer), BRCA1 (cancer), a-synuclein (PD, AD, dementia, Down syndrome), amyloidb(AD), tau (AD), prion(TSEs), amylin (Type II diabetes), hirudinand thrombin(CVD), HPV(cancer)…

  28. Human Papillomavirus (HPV) Increased amount of disorder in E6 proteins from high-risk HPVs Uversky et al, 2006, JPR, 5 (8), 1829-1842

  29. BRCA1 Mark et al, J Mol Biol. 2005, 345(2):275-87 CH plot of BRCA1 fragments BRCA1 fragments Full-length BRCA1 Disordered proteins CD and NMR of fragments- all disordered Ordered proteins Are disease proteins more disordered in general?

  30. Disorder and disease Disease-related SW keywords are strongly associated with predicted disorder (p>0.95) Xie et al, 2007, JPR

  31. Disease-associated mutations Disease mutations impact protein Structure: - Folding - Oligomerization - Stability - Activity … • Function: • Post-translational modifications • Binding to partners • Intracellular localization • … Many predictors of the functional impact of SNPs are available (SIFT, POLYPHEN, SNP3D etc) Majority rely on known protein 3D structure and evolutionary conservation

  32. Disease-associated mutations • Disordered regions: • Do not fold into 3D structure • Are generally less evolutionary conserved than ordered regions • Do current predictors make errors in predicting • impact of disease mutations in IDRs? Do disease mutations even occur in the regions of disorder?

  33. Disease-associated mutations Disease mutations are prevalent in ORDERED regions

  34. Disorder-to-Order transition p=1.06E-32 p=5.47E-105 Some disease mutations in disordered regions cause Disorder-> Order transition (may disrupt disordered structure? induce order?)

  35. D→O and O→D D→O O→D 32.2% 44%

  36. Arginine is often mutated

  37. Hypothetical mechanism? Codons for Arginine: CGG CGT CGC CGA AGA AGG TGG TGT TGC TGA AGA AGG R-> W R-> C R-> C R-> Stop N/A N/A CpGmethylation R-> W and R-> C are among the most frequent mutations in the disease dataset

  38. Disease Models Disorder-centric vs Structure-centric view at disease mutations

  39. IDRs summary • Proteins can carry intrinsically disordered • regions • These regions can be predicted from sequence • IDRs perform important functional roles: • PTMs, molecular recognition, involvement in • diseases • Disease mutations could occur in IDRs, and OR • and IDR mutations could lead to diseases via • different mechanisms

  40. Acknowledgements Rockefeller University: Jurg Ott Chad Haynes Fei Ji Columbia University: Vladimir Vacic PNNL: Eric Ackerman Richard D. Smith • Indiana University • Predrag Radivojac • Mark Goebl • Keith Dunker Funding:

  41. Disordered Proteins Database DisProt http://www.DisProt.org List of Disorder Predictors http://www.disprot.org/predictors.php Phos Sites Predictor DisPhos http://www.ist.temple.edu/disphos/ Ub Sites Predictor UbPred http://www.ubpred.org/ lilyak@ucsd.edu – Lilia Iakoucheva

  42. Prevalence of IDPs in nature % Sequences L > 40* % Sequences L > 40** Kingdom # Genomes 22 7 - 33 Bacteria 16 - 45 7 9 - 37 21 - 51 Archaea 5 35 - 51 52 - 67 Eukaryota * VL-XT Predictor, order ~ 78%, disorder ~ 65% ** VL2 Predictor, order ~ 83%, disorder ~ 75%

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