Editorial pubs.acs.org/CR
Introduction to Intrinsically Disordered Proteins (IDPs)
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or more than a century, molecular and structural biology, biochemistry, and protein biophysics have been dominated by a “rigid” or “semi-rigid” view of a functional protein molecule or functional protein domain. These functional entities were assumed to possess unique and stable 3D structures, a view supported by numerous reports of protein structures determined using X-ray crystallography and NMR spectroscopy (as of May 27, 2014, there were 97,857 structures of proteins and protein-nucleic acid complexes in the Protein Data Bank, with 87,536 of these structures (89.5%) determined by X-ray crystallography). These experimentally determined structures depicted protein molecules as aperiodic crystals, in which both atoms and backbone Ramachandran angles are relatively fixed and possess low-amplitude thermal fluctuations around their equilibrium positions. Although the functions of many proteins clearly fit within this structure−function paradigm, where a unique amino acid sequence encodes a unique energetically stable 3D fold associated with conformational fluctuations that allow for unique biological function, recent studies have revealed that many functional proteins or functional protein regions do not have unique 3D structures under functional conditions. In fact, contrarily to the ordered proteins and domains, such biologically active intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) have no single, well-defined equilibrium structure and exist as highly dynamic, heterogeneous ensembles of conformers resulting from their relatively flat free-energy surface. These IDPs/IDPRs are highly abundant in nature and have numerous biological activities. The number of structurally and functionally characterized IDPs and IDPRs is growing rapidly. The dramatic increase in corresponding scientific literature is the reflection of the growing interest in this class of proteins. This point is illustrated by Figure 1, which shows that, starting from the turn of the century, the number of papers dealing with the different aspects of intrinsically disordered proteins is exponentially increasing. The number of citations of these papers is also exponentially increasing at an exceeding rate, clearly indicating that protein disorder−related research is under increasing demand. Although Figure 1 seems to suggest that IDPs and IDPRs were discovered quite recently, the reality is more complex, and biologically active proteins without stable structures were rediscovered multiple times, showing that the phenomenon of biological functionality without stable structure has been periodically reported during the last 75 years or so. For a long time, this phenomenon was unnoticed by a wide audience because these highly dynamic proteins or protein regions have, over the years, been discovered one by one and described in the literature by a plethora of different names. In other words, this complex and lengthy route to recognizing these proteins as a novel class left in its path a trail of terms used for their description, giving rise to the intricate “paleontology” of this phenomenon. An incomplete list of terms used in the literature to describe these proteins includes floppy, pliable, rheomorphic,1 flexible,2 mobile,3 partially © 2014 American Chemical Society
Figure 1. Growing interest of researchers in intrinsically disordered proteins. Number of publications (red bars) and corresponding citations (blue bars) related to IDPs by year, from 1991 to 2014. Publications and citations were retrieved from a search of WEB of Science (http://apps.webofknowledge.com) using IDP-related terms: “(intrinsically OR natively OR inherently) AND (disordered OR unfolded OR unstructured OR flexible) AND (protein OR proteins)”. Inset represents accumulative citations in each year.
folded,4 natively denatured,5 natively unfolded,6,7 natively disordered,8 intrinsically unstructured,9,10 intrinsically denatured,5 intrinsically unfolded,6 intrinsically disordered,11 vulnerable,12 chameleon,13 malleable,14 4D,15 protein clouds,16 dancing proteins, 17 proteins waiting for partners, 18 3 2 proteins,13 and several other names that represent different combinations of “natively/naturally/inherently/intrinsically” with “unfolded/unstructured/disordered/denatured/flexible” among several other terms.19,20 The lack of common terminology was clearly the major reason precluding the appearance of the idea that this class of proteins constitutes a separate and important extension to the protein kingdom. The situation has changed at the turn of the century, mostly due to the bioinformatics studies that came to the important conclusion that naturally flexible proteins, instead of just being rare exceptions, represent a very broad class of proteins,7,9−11 and the unstructural biology came of age.21 The rapidly growing interest in IDPs can be attributed to several factors. The first of them is the role these proteins play in changing the understanding of the molecular mechanisms of protein action and in reshaping the protein structure−function relationship. The discovery of biologically active but extremely flexible proteins questioned the assumption that unique 3D structure is a prerequisite for protein function. Although IDPs lack stable structures at functional conditions, they are known to carry out a number of crucial biological functions that are complementary to the functional repertoire of structured (ordered) proteins. In any given organism, IDPs constitute a functionally broad and densely populated subset of its Special Issue: 2014 Intrinsically Disordered Proteins (IDPs) Published: July 9, 2014 6557
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Keith Dunker, Monika Fuxreiter, Julian Gough, Joerg Gsponer, David Jones, Philip Kim, Richard Kriwacki, Christopher Oldfield, Rohit Pappu, Peter Tompa, Vladimir Uversky, Peter Wright, and M. Madan Babu on classification of IDPs and IDPRs. These authors emphasize that structurally uncharacterized and currently nonannotated proteins and protein segments, which are commonly predicted to be disordered, represent a large source of functional novelty relevant for discovering new biology. The authors provide an important overview of the various classifications of IDPs and IDPRs that have been put forward in the literature and discuss diverse classification approaches based on function, functional elements, structure, sequence, protein interactions, evolution, regulation, and biophysical properties. They also suggest that combinations of multiple existing classification schemes provide a means to achieve high quality function prediction for IDPs and IDPRs, and potentially lead to the improved functional coverage and deeper understanding of protein function. The task of a precise structural description of IDPs is extremely difficult because of the insufficient independent experimental measurements compared to the number of degrees of conformational freedom. Among numerous techniques suitable for structural characterization of IDPs and IDPRs in solution, nuclear magnetic resonance (NMR) spectroscopy is considered as one of the most powerful experimental approaches for gaining structural information about these highly flexible entities at atomic resolution under conditions that are close to physiological. In their NMR-centric review, Malene Jensen, Markus Zweckstetter, Jie-rong Huang, and Martin Blackledge report on the recent progress in the interpretation of experimental NMR data to gain crucial information for accurate delineation of the conformational space sampled by IDPs/IDPRs in their free and bound forms. Peculiarities of protein structure and function are critically dependent on the environment. Even the most ordered proteins become unfolded under strong denaturing conditions. Many other environmental factors play crucial roles in controlling and regulating protein structure. A general property of every living organism is the complexity of its intracellular environment. In fact, proteins have evolved to function within cells, where the concentration of macromolecules, including proteins, nucleic acids, and carbohydrates within a cell can be as high as 400 g/L,22 creating a crowded medium, with considerably restricted amounts of free water.22−27 Obviously, “physiological conditions” commonly used in the majority of in vitro experiments, which are typically done at relatively ideal thermodynamic conditions of low protein and moderate salt concentrations, are a very poor model of the crowded cellular environment. Some crucial steps in making experimental environments more realistic include structural and functional analysis in the presence of model crowding agents, such as poly(ethylene glycol), dextran, Ficoll, inert proteins, etc.,28,29 or studying proteins directly inside the cell (e.g., by the in-cell NMR experiments that enable high-resolution investigations of proteins of interest directly in cellular environments30). Francois-Xavier Theillet, Andres Binolfi, Tamara FrembgenKesner, Karan Hingorani, Mohona Sarkar, Ciara Kyne, Conggang Li, Peter Crowley, Lila Gierasch, Gary Pielak, Adrian Elcock, Anne Gershenson, and Philipp Selenko analyze major physicochemical properties of cells and describe how IDPs might be affected by these various cellular properties. The next three reviews of this issue are dedicated to the careful analysis of some intricate aspects of disorder-based
proteome. The overall biological importance of IDPs/IDPRs, and their crucial roles in many biological processes, are further supported by the evolutionary persistence of these proteins and regions. IDPs are common across the three domains of life, being especially abundant in the eukaryotic proteomes. Signaling motifs and sites of posttranslational modifications are commonly located within IDPRs, and disorder-based signaling and functioning are modulated by alternative splicing. Second, IDPs are very attractive (and still poorly understood) subjects for theoretical and experimental characterization. For example, functional disorder-to-order transitions are very common in IDPs. Often, these transitions are coupled with the possibility that a single protein/region adopts different structures in complexes with different partners. Curiously, some ordered proteins require partial local unfolding and undergo order-to-disorder transitions to become functional, suggesting the existence of dormant functional disorder. The need for special means for the analysis of structural properties of IDPs/ IDPRs, their conformational behavior, their intrinsically flexible states, the mechanisms of their interactions with various binding partners, and their highly diversified functional roles in biological systems all create a foundation for the explosion in the development of novel experimental and theoretical tools and approaches for the analysis of IDPs. Biomedical aspects related to IDPs/IDPRs are also of great importance. In fact, intrinsic disorder is highly abundant among many proteins associated with various human diseases. Furthermore, IDPs are attractive drug targets and several small molecules have been shown to act by blocking protein− protein interactions that involve intrinsically disordered region of one of the partners. Although IDPs are major players in cell signaling, regulation, and recognition, and although they are frequently involved in the pathogenesis of numerous human diseases, the phenomenon of protein intrinsic disorder was not mentioned in the major Biochemistry textbooks until quite recently. The significant achievements in this area of biochemistry, molecular biology, structural biology, and biophysics have been presented in several reviews spread among dozens of scientific journals and books. The aim of this issue on Intrinsically Disordered Proteins is to introduce several important aspects of protein intrinsic disorder. Most of the reviews are written by teams comprising several authorities in the corresponding field and young scientists. This approach provides the multiangular consideration of a given subject and defines the comprehensive, authoritative, and critical nature of these reviews. First, Johnny Habchi, Peter Tompa, Sonia Longhi, and Vladimir Uversky have contributed a review that serves as a formal introduction to this thematic issue of Chemical Reviews on intrinsically disordered proteins. The authors start with a brief historical overview by showing the role of bioinformatics in establishing the IDP field. Then they provide a generalized description of the major computational and experimental tools which are used in the field for IDP analysis. They also discuss the variability of functional roles of IDPs, provide a brief analysis of the modes of IDP interactions with various binding partners, introduce the peculiarities of IDP evolution, discuss the abundance of IDPs in various proteomes, emphasize the roles of IDPs in the pathogenesis of various diseases, and stress the importance of IDPs as potential drug targets. The idea of a complex nature of “simple” disordered proteins is developed in the review by Robin van der Lee, Marija Buljan, Benjamin Lang, Robert Weatheritt, Gary W. Daughdrill, A. 6558
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cleoprotein complexes, scaffold proteins, cytoskeleton, and extracellular matrix. The review by Vladimir Uversky, Vrushank Davé, Lilia Iakoucheva, Prerna Malaney, Steven Metallo, Ravi Pathak, and Andreas Joerger considers different aspects of pathological unfoldomics and discusses the roles of intrinsically disordered proteins in the pathogenesis of human diseases. The authors emphasize that, since distortion of any of the mechanisms controlling IDP/IDPR functionality can be detrimental, IDPs/ IDPRs are commonly found in various human diseases, ranging from cancer to cardiovascular disease, to neurodegenerative diseases, to diabetes. The involvement of IDPs in pathology is commonly associated with some abnormalities in their regulation, where chromosome translocation, aberrant splicing, and alternative splicing, altered expression, abnormal posttranslational modifications, and pathological mutations all might play a role. Two important cancer-related proteins, p53 and PTEN, are then used as illustrative examples of pathogenic IDPs. The last part of this review is dedicated to the consideration of IDPs as potential drug targets and to the introduction of currently available approaches for finding small molecules affecting the functions of IDPs/IDPRs. The “IDPs in pathology” topic is continued by Bin Xue, David Blocquel, Johnny Habchi, Alexey Uversky, Lukasz Kurgan, Vladimir Uversky, and Sonia Longhi, who summarize the current knowledge on the abundance and roles of IDPs in viral proteomes. These authors discuss the unique origin and properties of viruses, provide a brief description of the classification of viral proteins, show which roles intrinsic disorder plays in structural proteins, focus on the multitude of functional roles of intrinsic disorder in nonstructural, regulatory, and accessory proteins, and discuss the role of intrinsic disorder in resolving potential structural chaos evoked by the use of common use alternative splicing and overlapping reading frames during the biosynthesis of viral proteins. The review by Macarena Marı ́n and Thomas Ott is dedicated to the abundance and roles of intrinsic disorder in plant proteins and phytopathogenic bacterial effectors. Among various aspects covered by this review are a general consideration of intrinsic disorder in plant proteins; IDPs involved in abiotic stress response and plant signaling; roles of disorder in chloroplast proteins; involvement of IDPs/IDPRs in plant immunity; and the roles of intrinsic disorder in effector proteins produced by plant pathogens, such as bacteria, fungi, oomycetes, and nematodes, to suppress or circumvent plant immune system. Finally, the review by Remy Loris and Abel Garcia-Pino discusses intricate dynamics-based regulatory mechanisms in toxin-antitoxin (TA) modules, which are small operons that encode two genes, a toxic protein, and antitoxin protecting cells from this toxin. It is stated that the TA modules are ubiquitous in the genomes of prokaryotes and archeae. Depending on the molecular mechanisms of their action, TA modules can be grouped into three major types. The review covers the modular organization and origin of these modules and shows that intrinsic disorder is common in TA antitoxins, where it plays various functional roles. In fact, TA modules use IDPRs for regulation at the level of protein activity and transcription and exemplify the variety of functionalities that can arise from simple folding-upon-binding interactions. In summary, this thematic issue provides a collection of focused articles in the field of intrinsically disordered proteins. These articles are not simply reviews, but they contain new
functionality. First, Peter Tompa extends the concept of allostery to IDPs. Originally, allostery was introduced as a regulatory mechanism, where the activity of a protein is modified or regulated by the binding of a ligand to a site distant/different from the active site, thereby allowing such a protein to serve as an allosteric switch reacting to a specific signal. Since IDPs/IDPRs are often characterized by a combination of multiple regulatory sites, they can integrate and interpret multiple incoming signals. The author also suggests that structural disorder contributes to multisteric regulation by modular signaling proteins which are built as a combination of domains, motifs, and linkers, and can display complex regulatory behavior. Such multistericity explains the long-range flow of regulatory information resulting from the remodeling of the conformational ensemble of complex proteins and perfectly fits into the signaling networks of higher eukaryotes. Next, Kim Van Roey, Bora Uyar, Robert Weatheritt, Holger Dinkel, Markus Seiler, Aidan Budd, Toby Gibson, and Norman Davey summarize the current state of the art in the field of short linear motifs, and they show that these ubiquitous and functionally diverse protein interaction modules are crucial functional elements of IDPs/IDPRs able to direct cell regulation. In the subsequent review, Ursula Jakob, Richard Kriwacki, and Vladimir Uversky introduce conditionally and transiently disordered proteins, which undergo environment- or modification-induced order-to-disorder transitions crucial for their function, and which are reversed back to their ordered, nonfunctional state as soon as the environment is restored or the modification is removed. In other words, such proteins possess cryptic or dormant functional disorder which needs to be awoken in order to make these proteins active. A wide spectrum of factors grouped into two major classes, passive (i.e., environmental factors that are not dependent on any specific interaction between the protein and its partner) and active (i.e., factors that involve some specific interaction of a protein with its environment), can induce functional order-todisorder transitions and activate corresponding proteins. Passive factors correspond to a modification of some global features of the protein environment, such as changes in pH, temperature, the redox potential, mechanical force, or light exposure, whereas active factors include interactions of a protein with membranes, ligands, other proteins, nucleic acids, or various post-translational modifications or release of autoinhibition. The various roles of intrinsic disorder in assembly and function of proteinaceous machines are considered in the review by Monika Fuxreiter, Á gnes Tóth-Petróczy, Daniel Kraut, Andreas Matouschek, Roderick Lim, Bin Xue, Lukasz Kurgan, and Vladimir Uversky. The authors start with the consideration of intrinsic disorder as a crucial factor for the assembly of protein complexes and show that ordered complexes can be formed from the disordered monomers, that the presence of intrinsic disorder in monomers provides a means for stepwise and directional assembly, that such disorder-controlled stepwise assembly can be dependent on binding to some hidden sites, and that binding-induced (partial) folding of an IDP can generate a new conformation with a novel binding site, thereby providing a means for binding chain reactions. These general concepts are then illustrated by some specific examples of pliable proteinaceous machines, such as mediator, protein unfolding machines, nucleopore, ribonu6559
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(4) Linderstrom-Lang, K.; Schellman, J. A. In The Enzymes, 2nd ed.; Boyer, P. D., Lardy, H., Myrback, K., Eds.; Academic Press: New York, 1959. (5) Schweers, O.; Schonbrunn-Hanebeck, E.; Marx, A.; Mandelkow, E. J. Biol. Chem. 1994, 269, 24290. (6) Weinreb, P. H.; Zhen, W.; Poon, A. W.; Conway, K. A.; Lansbury, P. T., Jr. Biochemistry 1996, 35, 13709. (7) Uversky, V. N.; Gillespie, J. R.; Fink, A. L. Proteins 2000, 41, 415. (8) Daughdrill, G. W.; Pielak, G. J.; Uversky, V. N.; Cortese, M. S.; Dunker, A. K. In Handbook of Protein Folding; Buchner, J., Kiefhaber, T., Eds.; Wiley-VCH, Verlag GmbH & Co. KGaA: Weinheim, Germany, 2005. (9) Wright, P. E.; Dyson, H. J. J. Mol. Biol. 1999, 293, 321. (10) Tompa, P. Trends Biochem. Sci. 2002, 27, 527. (11) Dunker, A. K.; Lawson, J. D.; Brown, C. J.; Williams, R. M.; Romero, P.; Oh, J. S.; Oldfield, C. J.; Campen, A. M.; Ratliff, C. M.; Hipps, K. W.; Ausio, J.; Nissen, M. S.; Reeves, R.; Kang, C.; Kissinger, C. R.; Bailey, R. W.; Griswold, M. D.; Chiu, W.; Garner, E. C.; Obradovic, Z. J. Mol. Graph. Model. 2001, 19, 26. (12) Chen, J.; Liang, H.; Fernandez, A. Genome Biol. 2008, 9, R107. (13) Uversky, V. N. J. Biomol. Struct. Dyn. 2003, 21, 211. (14) Fuxreiter, M.; Tompa, P.; Simon, I.; Uversky, V. N.; Hansen, J. C.; Asturias, F. J. Nat. Chem. Biol. 2008, 4, 728. (15) Tsvetkov, P.; Asher, G.; Paz, A.; Reuven, N.; Sussman, J. L.; Silman, I.; Shaul, Y. Proteins 2008, 70, 1357. (16) Dunker, A. K.; Uversky, V. N. Curr. Opin. Pharmacol. 2010, 10, 782. (17) Livesay, D. R. Curr. Opin. Pharmacol. 2010, 10, 706. (18) Janin, J.; Sternberg, M. J. E. F1000 Biol. Rep. 2013, 5, 2. (19) Dunker, A. K.; Babu, M. M.; Barbar, E.; Blackledge, M.; Bondos, S. E.; Dosztányi, Z.; Dyson, H. J.; Forman-Kay, J.; Fuxreiter, M.; Gsponer, J.; Han, K.-H.; Jones, D. T.; Longhi, S.; Metallo, S. J.; Nishikawa, K.; Nussinov, R.; Obradovic, Z.; Pappu, R.; Rost, B.; Selenko, P.; Subramaniam, V.; Sussman, J. L.; Tompa, P.; Uversky, V. N. Intrinsically Disordered Proteins 2013, 1, e24157. (20) Uversky, V. N.; Dunker, A. K. Biochim. Biophys. Acta 2010, 1804, 1231. (21) Tompa, P. Curr. Opin. Struct. Biol. 2011, 21, 419. (22) Zimmerman, S. B.; Trach, S. O. J. Mol. Biol. 1991, 222, 599. (23) Zimmerman, S. B.; Minton, A. P. Annu. Rev. Biophys. Biomol. Struct. 1993, 22, 27. (24) Fulton, A. B. Cell 1982, 30, 345. (25) Minton, A. P. Curr. Opin. Biotechnol. 1997, 8, 65. (26) Ellis, R. J. Trends Biochem. Sci. 2001, 26, 597. (27) Minton, A. P. Curr. Biol. 2000, 10, R97. (28) Minton, A. P. J. Biol. Chem. 2001, 276, 10577. (29) Hatters, D. M.; Minton, A. P.; Howlett, G. J. J. Biol. Chem. 2002, 277, 7824. (30) Ito, Y.; Selenko, P. Curr. Opin. Struct. Biol. 2010, 20, 640.
interpretations and opinions of authors. Obviously, not all important aspects are covered, and this collection of reviews is meant to serve as a starting point for future discussions of this intriguing phenomenon. I hope that the conclusions and opinions collected in this thematic issue will serve as promoters of future research. Finally, I would like to acknowledge all the authors who contributed their time and put a lot of effort into bringing this project to fruition. I am also thankful to Prof. Robert Kuchta for the invitation to serve as the Guest Editor for this thematic issue. I am particularly indebted to Saundra Richter for her invaluable help at different stages of this project.
Vladimir N. Uversky* University of South Florida King Abdulaziz University Russian Academy of Sciences
AUTHOR INFORMATION Corresponding Author
*Phone: 1-813-974-5816; Fax: 1-813-974-7357; E-mail:
[email protected]. Notes
Views expressed in this editorial are those of the author and not necessarily the views of the ACS. Biography
Vladimir Uversky obtained his Ph.D. in biophysics from Moscow Institute of Physics and Technology (1991) and his D.Sc. in biophysics from the Institute of Experimental and Theoretical Biophysics, Russian Academy of Sciences (1998). He spent his early career working on protein folding at the Institute of Protein Research and the Institute for Biological Instrumentation (Russian Academy of Sciences). In 1998, he moved to the University of California, Santa Cruz, to work on protein folding and misfolding and protein intrinsic disorder. In 2004, he moved to the Center for Computational Biology and Bioinformatics at Indiana UniversityPurdue University Indianapolis to work on intrinsically disordered proteins. Since 2010, he has been with the Department of Molecular Biology at the University of South Florida.
REFERENCES (1) Holt, C.; Sawyer, L. J. Chem. Soc., Faraday Trans. 1993, 89, 2683. (2) Pullen, R. A.; Jenkins, J. A.; Tickle, I. J.; Wood, S. P.; Blundell, T. L. Mol. Cell. Biochem. 1975, 8, 5. (3) Cary, P. D.; Moss, T.; Bradbury, E. M. Eur. J. Biochem. 1978, 89, 475. 6560
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