Intrinsically Disordered Protein: Difference between revisions

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<ref>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 [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-lecexture.html Anfinsen's Nobel Lecture.]</ref><ref>A similar observation was made around the same time by then graduate student Lisa Steiner in the lab of [[Richards, Frederic M.|Fred Richards]] at Yale University. Neither Richards nor advisor Joseph Fruton thought the observation interesting enough to publish. It was an answer to a question not yet asked. This story is recounted by David Eisenberg, see the next citation.</ref><ref>PMID: 29958112</ref>. They concluded that the amino acid sequence is sufficient for a protein to fold into its functional, lowest energy conformation. This work won the [[Nobel_Prizes_for_3D_Molecular_Structure|1972 Nobel Prize]], and was subsequently confirmed and extended by many researchers.

 

 

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<ref name="tompa2002" />. 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.

 

 

 

At left is an animation of a heat shock/chaperonin protein fragment. Residues 1-70 are disordered; 71-109 are alpha helical. This animates 20 models from an NMR experiment ([[2ljl]]). Charges are colored <font color="blue">'''blue=positive'''</font> and <font color="red">'''red=negative'''</font>. A high charge density prevents folding. For comparison, at right is an animation of 20 NMR models of a protein of similar length that folds into a stable domain ([[2n5a]]).

 

 

 

 

Animations will stop after 25 cycles. Shift+Re-load this page to restart the animations. ''Internet Explorer'' and ''Edge'': toggle spinning off to speed up animation. Click on an animation to see it larger.

 

 

Many [[X-ray crystallography|crystallographic]] structures have missing loops -- that is, ranges of amino acids with no [[atomic coordinate file|atomic coordinates]] in the model. These &quot;gaps&quot; 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<ref name="IDSG" />. Such gaps are the basis for the [[#Protein disorder predictors|DISOPRED2 disorder prediction server]]. [[FirstGlance in Jmol]] offers [[Temperature_value#Missing_Residues|one method for locating and visualizing such gaps]].

 

Despite the existence of compelling evidence for IDPs and intrinsically disordered loops beginning in 1990<ref name="struhl1990" /><ref>PMID: 2236048</ref><ref>For the ''unstructured domain'' interpretation of early work by Pontius and Berg, see the 2004 review by Tompa and Csermley, PMID: 15284216</ref>, many current textbooks of biochemistry and even some monographs on protein structure fail to mention intrinsic disorder and its importance for protein function<ref>PMID: 18831774</ref><ref>Martz, E. Book review of <i>Introduction to protein science—architecture, function, and genomics: Lesk, Arthur M.</i>. <i>Biochem. Mol. Biol. Educ.</i> 33:144-5 (2006). [http://dx.doi.org/10.1002/bmb.2005.494033022442 DOI: 10.1002/bmb.2005.494033022442]</ref>. In 2011, Chouard provided a readable and informative overview of IDPs and how some of them function<ref>PMID: 21390105</ref>.

== Examples of IDPs ==

Examples cover a wide variety of cellular systems and it has been predicted that eukaryotes have more IDPs than other kingdoms <ref>PMID: 11700597</ref>. Of course, there are no [[PDB codes]] for fully disordered proteins in isolation. However, there are some crystallographic results for IDP that undergo [[#Many IDPs undergo disorder-order transition|disorder-order transition]] when they complex with another folded protein domain, such as [[1jsu]], [[1g3j]], and [[1oct]]<ref name="tompa2002" />. Other examples are at [[Globular_Proteins]]. See further information about [[1jsu]] and other cases [[#Many IDPs undergo disorder-order transition|below]].

IDPs play roles in '''processes''' such as:

* DNA recognition molecules, e.g. the basic DNA-binding region of the leucine zipper protein, GCN4<ref name="struhl1990">PMID: 2145515</ref>

* Protein-RNA recognition, e.g. ribosomal proteins, such as L11-C76<ref>PMID: 8989327</ref> and [[Large Ribosomal Subunit of Haloarcula#The proteins:|several others]]<ref>PMID:10937989</ref>

 

* Transcriptional activation domains, e.g. NF-kb<ref>PMID: 7929265</ref>, Glucocorticoid receptor, 77-262 fragment <ref>PMID: 10196139</ref>. There is "widespread importance of structural disorder in gene regulatory proteins", such as Lacl/GalR and Hox<ref>PMID: 26342073</ref>.

[[Image:FoldIndex.jpeg | thumb | FoldIndex<ref name="foldindex">PMID: 15955783</ref> 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]]</td><td>[[Image:DisorderAA.jpg | thumb | 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 IDP (gray) <ref>PMID: 14579366</ref>]]

* [http://d2p2.pro/ D²P²]: "pre-computed disorder predictions on a large library of proteins from completely-sequenced genomes. ... statistical comparisons of the various prediction methods ...."

* [http://bioinf.cs.ucl.ac.uk/disopred/ DISOPRED2] (Jones Group, University College London, UK). "DISOPRED2 was trained on a set of around 750 non-redundant sequences with high resolution X-ray structures. Disorder was identified with those residues that appear in the sequence records but with coordinates missing from the electron density map. This is an imperfect means for identifying disordered residues as missing co-ordinates can also arise as an artifact of the crystalization process. False assignment of order can also occur as a result of stabilizing interactions by ligands or other macromolecules in the complex. However, this is the simplest means for defining disorder in the absence of further experimental investigation of the protein." (Quoted from the DISOPRED2 website.)

* [http://biomine.cs.vcu.edu/servers/flDPnn2/ flDPnn2] (putative '''f'''unction- and '''l'''inker based '''D'''isorder '''P'''rediction using deep '''n'''eural '''n'''etwork)<ref name="fldpnn">PMID: 34290238</ref>. In 2021, flDPnn, was selected as the {{font color|#c000c0|'''best disorder predictor in the first Critical Assessment of Protein Intrinsic Disorder Prediction'''}} (CAID) <ref name="caid2021">PMID: 33875885</ref>.

 

* [https://fold.proteopedia.org/ FoldIndex]<ref name="foldindex" /> (Sussman Group, Weizmann Institute, Rehovot, Israel). FoldIndex makes predictions based on the observation that IDPs occupy the low hydrophobicity/ high net-charge portion of charge-hydrophobicity phase space. (See Figure above.)

 

* [https://iupred2a.elte.hu/ IUPred2a] (Dosztányi, Csizmók, Tompa and Simon: Budapest, Hungary). "IUPred recognized intrinsically unstructured regions from the amino acid sequence based on the estimated pairwise energy content. The underlying assumption is that globular proteins are composed of amino acids which have the potential to form a large number of favorable interactions, whereas intrinsically disorered proteins (IDPs) adopt no stable structure because their amino acid composition does not allow sufficient favorable interactions to form." (Quoted from the IUPred website.)

 

* [http://www.pondr.com/ PONDR] (Dunker Group, Indiana University and Molecular Kinetics, Inc., Indianapolis IN USA; Obradovic Group, Temple Univ., Philadelphia PA USA). "PONDR® functions from primary sequence data alone. The predictors are feedforward neural networks that use sequence information from windows of generally 21 amino acids. Attributes, such as the fractional composition of particular amino acids or hydropathy, are calculated over this window, and these values are used as inputs for the predictor. The neural network, which has been trained on a specific set of ordered and disordered sequences, then outputs a value for the central amino acid in the window. The predictions are then smoothed over a sliding window of 9 amino acids. If a residue value exceeds a threshold of 0.5 (the threshold used for training) the residue is considered disordered." (Quoted from the PONDR website.)

 

* [https://app.strubi.ox.ac.uk/RONN/ RONN] (Esnouf Group, University of Oxford, UK). "We have developed the regional order neural network (RONN) software as an application of our recently developed ‘bio- basis function neural network’ pattern recognition algorithm to the detection of natively disordered regions in proteins. The results of blind-testing a panel of nine disorder prediction tools (including RONN) against 80 protein sequences derived from the Protein Data Bank shows that, based on the probability excess measure, RONN performed the best."<ref>PMID: 15947016</ref>

 

* [http://prodata.swmed.edu/Lab/Software.htm WinDiso]<ref>PMID: 17893360</ref> (Grishin Lab, Dallas, Texas USA). "WinDiso is a linear, sequence- and alignment-based predictor of disordered/unfolded regions in proteins. It has the capability of adjusting for the increased tendency for disorder at protein termini. The simple weighted window-based algorithm and careful optimization technique make this a good predictor to use when trying to avoid bias toward special cases." (Quoted from the Grishin lab website.)

 

''The above list is incomplete. Addition of other servers is welcome, and summaries of methods, pros and cons for each server would be useful.''

 

 

 

Because their very nature makes them difficult to categorize and study by standard means, several groups have set up curated listings of intrinsically disordered proteins and intrinsically disordered regions.

 

 

* [http://pedb.vib.be/ PED: Protein Ensemble Database]: " an openly accessible database for the deposition of structural ensembles of intrinsically disordered proteins (IDPs) and of denatured proteins based on nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and other data measured in solution." The database of conformational ensembles describing flexible proteins. This database seems to have stopped taking submissions in 2016.

 

 

* [http://www.disprot.org/ Disprot]: " a database of experimental evidences of disorder manually collected from literature."

 

 

 

== Evolution of IDPs ==

In p53, the folded DNA-binding domain is conserved, while the intrinsically disordered regions display a higher rate of mutations<ref>PMID: 23352836</ref>.

 

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 p27^Kip1. p27^Kip1 is an IDP and it binds to phosphorylated <scene name='User:Tzviya_Zeev-Ben-Mordehai/Sandbox_1/Complex/2'>cyclin/CDK complex</scene> in <scene name='User:Tzviya_Zeev-Ben-Mordehai/Sandbox_1/Extended/2'>an extended conformation</scene> interacting with both <scene name='User:Tzviya_Zeev-Ben-Mordehai/Sandbox_1/Cyca/2'>cyclin A</scene> and <scene name='User:Tzviya_Zeev-Ben-Mordehai/Sandbox_1/Cdk2/3'>CDK2</scene> ([[1jsu]]). 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. [[http://www.proteopedia.org/wiki/index.php/1jsu]]

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