Proteopedia

Intrinsically Disordered Protein

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It has long been taught that proteins must be properly folded in order to perform their functions. This paradigm derives from work by Christian B. Anfinsen and coworkers. In the 1960's, they showed that RNAse, when denatured so that 99% of its enzymatic activity was lost, could regain enzymatic activity within seconds when the denaturing agent was removed under proper conditions[1]. They concluded that the amino acid sequence is sufficient for a protein to fold into its functional, lowest energy conformation. This work won the 1972 Nobel Prize, and was subsequently confirmed and extended by many researchers.

Recently, it has been recognized that not all proteins function in a folded state[2][3][4][5][6]. Some proteins must be unfolded or disordered in order to perform their functions, and others fold only in complex with target structures[7][8][9]. These are termed intrinsically disordered protein (IDP), intrinsically unstructured protein (IUP), or natively unfolded protein.

By some estimates, about 10% of all proteins are fully disordered, and about 40% of eukaryotic proteins have at least one long (>50 amino acids) disordered loop[5]. Such sequences, under physiological conditions in vitro, display physicochemical characteristics resembling those of random coils. They possess little or no ordered structure, having instead an extended conformation with high intra-molecular flexibility, lacking any tightly packed core.

Many crystallographic structures have missing loops -- that is, ranges of amino acids with no atomic coordinates in the model. These "gaps" in the model are often thought to be artifacts of inadvertant disorder in the crystal. In some cases, these gaps may be alerting us to the presence of intrinsically disordered loops in an otherwise folded protein. FirstGlance in Jmol offers one method for locating such gaps.

Examples of IUPsExamples of IUPs

Examples cover a wide variety of cellular systems and it has been predicted that eukaryotes have more IUPs than other kingdoms [10]. Of course, there are no PDB codes for fully disordered proteins in isolation. However, there are some crystallographic results for IUP that fold in complex with another folded protein domain, such as 1jsu, 1g3j, and 1oct[5]. See further information about 1jsu and another case below.

IUPs play roles in processes such as:

  • Cell signaling and cell cycle regulation, e.g. cyclin dependent kinase inhibitor p21Waf1/Cip1/Sdi1[11]
  • Oncogene, e.g. P53 contains large unstructured regions in its native state[12]
  • Assembly of cytoskeletal proteins, e.g. Tau protein[13]
  • Membrane fusion and membrane transport, e.g. isolated components of the SNARE complex[14]
  • DNA recognition molecules, e.g. the basic DNA-binding region of the leucine zipper protein, GCN4[15]
  • Protein-RNA recognition, e.g. ribosomal proteins L11-C76[16]
  • Transcriptional activation domains, e.g. NF-kb[17], Glucocorticoid receptor, 77-262 fragment [18]
  • Amyloid formation, e.g. prion protein, N terminal part[19], NACP precursor of the non-Ab component of the amyloid plaque[20]

Protein disorder predictorsProtein disorder predictors

 
FoldIndex[21] output for three protein sequences (a) Cat-Muscle Pyruvate Kinase (b) The human p53 tumor suppressor protein (c) Chicken gizzard caldesmon; green is folded and red is unfolded
 
Content of order-promoting and disorder-promoting amino acids in the Drosophila proteome (black) and in the cytoplasmic domain of gliotactin that was shown to be IUP (gray) [22]

Led by the assumption that “since amino acid sequence determines 3-D structure, amino acid sequence should also determine lack of 3-D structure” [23] specific sequence features shared by IUPs have been evaluated and algorithms for their identification formulated.


IUPs occupy the low hydrophobicity/ high net-charge portion of charge-hydrophobicity phase space, which is the basis of the FoldIndex predictor .

Other predictors includes, DISOPRED, PONDR, IUPred and more.

The low hydrophobicity and high net charge of naively unfolded proteins result in a difference in amino acid composition between them and naively folded proteins [24]. Compared to sequences of ordered proteins, disordered protein sequences are substantially depleted in I, L, V, W, F, Y, and C, which were therefore designated as “order promoting” amino acids, and enriched in E, K, R, G, Q, S, P, and A, which have been designated as “disorder promoting”. The under representation of hydrophobic amino acids in a protein diminishes one of the basic thermodynamic forces known to be important for protein folding, namely, the hydrophobic interaction. Because a hydrophobic core does not form, such proteins have large hydrodynamic dimensions.

Biological implications of IUPsBiological implications of IUPs

It was proposed that the unfolded nature of the IUPs provides them with advantages in recognition and binding. Although their large hydrodynamic dimensions slow down diffusion, their size provides a large target for initial molecular collisions, and the lack of rigid binding pockets permits multiple approach orientations for a binding partner, which may increase the probability of productive interactions [25][23]. In addition, IUPs allow molecular plasticity by adopting more than one conformation and binding diversity by binding to several proteins and thus many of the known hub proteins are IUPs. IUPs rapid turnover in the cell allow their tight regulation as many times needed in cell signaling and cell cycle.

Many IUPs undergo disorder-order transitionMany IUPs undergo disorder-order transition

Binding of natural ligands such as a variety of small molecules, substrates, cofactors, other proteins, nucleic acids or membranes induces folding of unstructured proteins. See the following two examples and also the page on Lac repressor

The human p27Kip1 kinase inhibitory domain [26]The human p27Kip1 kinase inhibitory domain [26]

The cyclin-dependent kinases (CDKs) have a central role in coordinating the eukaryotic cell division cycle. CDKs are controlled through several different processes involving the binding of activating cyclin subunits. Complexes of cyclins with CDKs play a central role in the control of the eukaryotic cell cycle. These complexes are inhibited by other proteins termed in general cyclin-CDK inhibitors (CKIs). One example of CKIs is p27Kip1. p27Kip1 is an IUP and it binds to phosphorylated in interacting with both and . On cyclin A, it binds in a groove formed by conserved cyclin box residues. On CDK2, it binds and rearranges the amino-terminal lobe and also inserts into the catalytic cleft, mimicking ATP. [[1]]

Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex (1jsu)

Drag the structure with the mouse to rotate

The transcriptional activator GCN4 [27]The transcriptional activator GCN4 [27]

The yeast transcriptional activator GCN4 belongs to a large family of eukaryotic transcription factors including Fos, Jun and CREB. All family members have a DNA recognition motif consists of a coiled-coil dimerization element, the leucine-zipper, and an adjoining basic region, which mediates DNA binding. This basic region is largely unstructured in the absence of DNA, addition of DNA containing a GCN4 binding site induce the transition of this region from unstructured to α-helical.

File:1dgc d.png
The structure of GCN4 bound to a DNA fragment (1dgc) containing the perfectly symmetrical binding site. A homodimer of parallel alpha-helices form an interhelix coiled-coil region via the leucine zipper, and the two N-terminal basic regions fit into the major groove of half sites on opposite sides of the DNA double helix. Arginine and Lysines shown in sticks representation.

References and NotesReferences and Notes

  1. ↑ For the sake of brevity, this description is oversimplified. RNAse needed to be reduced to break disulfide bonds, as well as using 8 M urea, for denaturation. Oxidation without the denaturant then left an inactive enzyme because the disulfide bonds formed randomly, precluding proper folding except very slowly (many hours). Only when protein disulfide isomerase was added did the re-folding occur at a physiological rate (about a minute). The fact that RNAse could thus be trapped in an inactive conformation under physiological conditions contributed to the insights developed by Anfinsen and his team. Proteins lacking disulfides renatured in seconds. For details, see Anfinsen's Nobel Lecture.
  2. ↑ Wright PE, Dyson HJ. Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. J Mol Biol. 1999 Oct 22;293(2):321-31. PMID:10550212 doi:10.1006/jmbi.1999.3110
  3. ↑ Dunker AK, Lawson JD, Brown CJ, Williams RM, Romero P, Oh JS, Oldfield CJ, Campen AM, Ratliff CM, Hipps KW, Ausio J, Nissen MS, Reeves R, Kang C, Kissinger CR, Bailey RW, Griswold MD, Chiu W, Garner EC, Obradovic Z. Intrinsically disordered protein. J Mol Graph Model. 2001;19(1):26-59. PMID:11381529
  4. ↑ Uversky VN. What does it mean to be natively unfolded? Eur J Biochem. 2002 Jan;269(1):2-12. PMID:11784292
  5. ↑ 5.0 5.1 5.2 Tompa P. Intrinsically unstructured proteins. Trends Biochem Sci. 2002 Oct;27(10):527-33. PMID:12368089
  6. ↑ Summary of the previous paper (Tompa, 2002): The disorder of intrinsically unstructured proteins (IUP's) is crucial to their functions. They may adopt defined but extended structures when bound to cognate ligands. Their amino acid compositions are less hydrophobic than those of soluble proteins. They lack hydrophobic cores, and hence do not become insoluble when heated. About 40% of eukaryotic proteins have at least one long (>50 residues) disordered region. Roughly 10% of proteins in various genomes have been predicted to be fully disordered. Presently over 100 IUP's have been identified; none are enzymes. Obviously, IUP's are greatly underrepresented in the Protein Data Bank, although there are a few cases of an IUP bound to a folded (intrinsically structured) protein. Here, Tompa suggests five functional categories for intrinsically unstructured proteins and domains: entropic chains (bristles to ensure spacing, springs, flexible spacers/linkers), effectors (inhibitors and disassemblers), scavengers, assemblers, and display sites. (Summary by Eric Martz.)
  7. ↑ Gunasekaran K, Tsai CJ, Kumar S, Zanuy D, Nussinov R. Extended disordered proteins: targeting function with less scaffold. Trends Biochem Sci. 2003 Feb;28(2):81-5. PMID:12575995
  8. ↑ Summary of the previous paper (Gunasekaran et al., 2003): Argues that proteins involved in extensive protein-protein interactions can function effectively despite having their structure depend upon such interactions, so that as monomers they are natively disordered. Dispensing with the structural framework (scaffold) needed to maintain a stable fold in the monomer increases efficiency by reducing size. This may account for the large percentage (roughly half) of all proteins that are predicted to be natively disordered. (Summary by Eric Martz.)
  9. ↑ Dyson HJ, Wright PE. Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol. 2005 Mar;6(3):197-208. PMID:15738986 doi:10.1038/nrm1589
  10. ↑ Dunker AK, Obradovic Z, Romero P, Garner EC, Brown CJ. Intrinsic protein disorder in complete genomes. Genome Inform Ser Workshop Genome Inform. 2000;11:161-71. PMID:11700597
  11. ↑ Kriwacki RW, Hengst L, Tennant L, Reed SI, Wright PE. 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. 1996 Oct 15;93(21):11504-9. PMID:8876165
  12. ↑ Bell S, Klein C, Muller L, Hansen S, Buchner J. p53 contains large unstructured regions in its native state. J Mol Biol. 2002 Oct 4;322(5):917-27. PMID:12367518
  13. ↑ Schweers O, Schonbrunn-Hanebeck E, Marx A, Mandelkow E. Structural studies of tau protein and Alzheimer paired helical filaments show no evidence for beta-structure. J Biol Chem. 1994 Sep 30;269(39):24290-7. PMID:7929085
  14. ↑ Fiebig KM, Rice LM, Pollock E, Brunger AT. Folding intermediates of SNARE complex assembly. Nat Struct Biol. 1999 Feb;6(2):117-23. PMID:10048921 doi:10.1038/5803
  15. ↑ Weiss MA, Ellenberger T, Wobbe CR, Lee JP, Harrison SC, Struhl K. Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA. Nature. 1990 Oct 11;347(6293):575-8. PMID:2145515 doi:http://dx.doi.org/10.1038/347575a0
  16. ↑ Markus MA, Hinck AP, Huang S, Draper DE, Torchia DA. High resolution solution structure of ribosomal protein L11-C76, a helical protein with a flexible loop that becomes structured upon binding to RNA. Nat Struct Biol. 1997 Jan;4(1):70-7. PMID:8989327
  17. ↑ Schmitz ML, dos Santos Silva MA, Altmann H, Czisch M, Holak TA, Baeuerle PA. Structural and functional analysis of the NF-kappa B p65 C terminus. An acidic and modular transactivation domain with the potential to adopt an alpha-helical conformation. J Biol Chem. 1994 Oct 14;269(41):25613-20. PMID:7929265
  18. ↑ Baskakov IV, Kumar R, Srinivasan G, Ji YS, Bolen DW, Thompson EB. Trimethylamine N-oxide-induced cooperative folding of an intrinsically unfolded transcription-activating fragment of human glucocorticoid receptor. J Biol Chem. 1999 Apr 16;274(16):10693-6. PMID:10196139
  19. ↑ Donne DG, Viles JH, Groth D, Mehlhorn I, James TL, Cohen FE, Prusiner SB, Wright PE, Dyson HJ. 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. 1997 Dec 9;94(25):13452-7. PMID:9391046
  20. ↑ Weinreb PH, Zhen W, Poon AW, Conway KA, Lansbury PT Jr. NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded. Biochemistry. 1996 Oct 29;35(43):13709-15. PMID:8901511 doi:10.1021/bi961799n
  21. ↑ Prilusky J, Felder CE, Zeev-Ben-Mordehai T, Rydberg EH, Man O, Beckmann JS, Silman I, Sussman JL. FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics. 2005 Aug 15;21(16):3435-8. Epub 2005 Jun 14. PMID:15955783 doi:http://dx.doi.org/10.1093/bioinformatics/bti537
  22. ↑ Zeev-Ben-Mordehai T, Rydberg EH, Solomon A, Toker L, Auld VJ, Silman I, Botti S, Sussman JL. The intracellular domain of the Drosophila cholinesterase-like neural adhesion protein, gliotactin, is natively unfolded. Proteins. 2003 Nov 15;53(3):758-67. PMID:14579366 doi:10.1002/prot.10471
  23. ↑ 23.0 23.1 Dunker AK, Obradovic Z. The protein trinity--linking function and disorder. Nat Biotechnol. 2001 Sep;19(9):805-6. PMID:11533628 doi:10.1038/nbt0901-805
  24. ↑ Romero P, Obradovic Z, Li X, Garner EC, Brown CJ, Dunker AK. Sequence complexity of disordered protein. Proteins. 2001 Jan 1;42(1):38-48. PMID:11093259
  25. ↑ Denning DP, Uversky V, Patel SS, Fink AL, Rexach M. The Saccharomyces cerevisiae nucleoporin Nup2p is a natively unfolded protein. J Biol Chem. 2002 Sep 6;277(36):33447-55. Epub 2002 Jun 13. PMID:12065587 doi:10.1074/jbc.M203499200
  26. ↑ Russo AA, Jeffrey PD, Patten AK, Massague J, Pavletich NP. Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature. 1996 Jul 25;382(6589):325-31. PMID:8684460 doi:10.1038/382325a0
  27. ↑ Konig P, Richmond TJ. The X-ray structure of the GCN4-bZIP bound to ATF/CREB site DNA shows the complex depends on DNA flexibility. J Mol Biol. 1993 Sep 5;233(1):139-54. PMID:8377181 doi:http://dx.doi.org/10.1006/jmbi.1993.1490

AuthorshipAuthorship

This article was written by Tzviya Zeev-Ben-Mordehai. Contributions by Eric Martz were minor -- his name is listed first due to a technicality.

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

Tzviya Zeev-Ben-Mordehai, Eric Martz, Jaime Prilusky, Eran Hodis, Wayne Decatur, Joel L. Sussman, Karl Oberholser, David Canner, Alexander Berchansky, Michal Harel
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