Abstract
Intrinsically disordered protein regions exist in a collection of dynamic interconverting conformations that lack a stable 3D structure. These regions are structurally heterogeneous, ubiquitous and found across all kingdoms of life. Despite the absence of a defined 3D structure, disordered regions are essential for cellular processes ranging from transcriptional control and cell signalling to subcellular organization. Through their conformational malleability and adaptability, disordered regions extend the repertoire of macromolecular interactions and are readily tunable by their structural and chemical context, making them ideal responders to regulatory cues. Recent work has led to major advances in understanding the link between protein sequence and conformational behaviour in disordered regions, yet the link between sequence and molecular function is less well defined. Here we consider the biochemical and biophysical foundations that underlie how and why disordered regions can engage in productive cellular functions, provide examples of emerging concepts and discuss how protein disorder contributes to intracellular information processing and regulation of cellular function.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Dunker, A. K. et al. Intrinsically disordered protein. J. Mol. Graph. Model. 19, 26–59 (2001). Together with Wright and Dyson (1999), Uversky (2002) and Tompa (2002), this article makes the original arguments that IDRs can and do have important roles in cellular function.
Wright, P. E. & Dyson, H. J. Intrinsically unstructured proteins: re-assessing the protein structure–function paradigm. J. Mol. Biol. 293, 321–331 (1999).
van der Lee, R. et al. Classification of intrinsically disordered regions and proteins. Chem. Rev. 114, 6589–6631 (2014).
Uversky, V. N. Natively unfolded proteins: a point where biology waits for physics. Protein Sci. 11, 739–756 (2002).
Tompa, P. Intrinsically unstructured proteins. Trends Biochem. Sci. 27, 527–533 (2002).
Brodsky, S., Jana, T. & Barkai, N. Order through disorder: the role of intrinsically disordered regions in transcription factor binding specificity. Curr. Opin. Struct. Biol. 71, 110–115 (2021).
Fuxreiter, M. et al. Malleable machines take shape in eukaryotic transcriptional regulation. Nat. Chem. Biol. 4, 728–737 (2008).
Zhang, X., Bai, X.-C. & Chen, Z. J. Structures and mechanisms in the cGAS–STING innate immunity pathway. Immunity 53, 43–53 (2020).
Cuylen, S. et al. Ki-67 acts as a biological surfactant to disperse mitotic chromosomes. Nature 535, 308–312 (2016).
Pelham, J. F., Dunlap, J. C. & Hurley, J. M. Intrinsic disorder is an essential characteristic of components in the conserved circadian circuit. Cell Commun. Signal. 18, 181 (2020).
Tompa, P. & Csermely, P. The role of structural disorder in the function of RNA and protein chaperones. FASEB J. 18, 1169–1175 (2004).
Altmeyer, M. et al. Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose). Nat. Commun. 6, 8088 (2015).
Wright, P. E. & Dyson, H. J. Intrinsically disordered proteins in cellular signalling and regulation. Nat. Rev. Mol. Cell Biol. 16, 18–29 (2014).
Payliss, B. J. et al. Phosphorylation of the DNA repair scaffold SLX4 drives folding of the SAP domain and activation of the MUS81-EME1 endonuclease. Cell Rep. 41, 111537 (2022).
Witus, S. R. et al. BRCA1/BARD1 intrinsically disordered regions facilitate chromatin recruitment and ubiquitylation. EMBO J. 42, e113565 (2023).
Yanez Orozco, I. S. et al. Identifying weak interdomain interactions that stabilize the supertertiary structure of the N-terminal tandem PDZ domains of PSD-95. Nat. Commun. 9, 3724 (2018).
Watson, M. et al. Hidden multivalency in phosphatase recruitment by a disordered AKAP scaffold. J. Mol. Biol. 434, 167682 (2022).
Riback, J. A. et al. Stress-triggered phase separation is an adaptive, evolutionarily tuned response. Cell 168, 1028–1040.e19 (2017).
Feric, M. et al. Coexisting liquid phases underlie nucleolar subcompartments. Cell 165, 1686–1697 (2016).
Cermakova, K. et al. A ubiquitous disordered protein interaction module orchestrates transcription elongation. Science 374, 1113–1121 (2021).
Borcherds, W. et al. Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells. Nat. Chem. Biol. 10, 1000–1002 (2014). This paper offers direct evidence that ensemble properties of IDRs can directly influence cellular function.
Dyla, M. & Kjaergaard, M. Intrinsically disordered linkers control tethered kinases via effective concentration. Proc. Natl Acad. Sci. USA 117, 21413–21419 (2020).
Millard, P. S. et al. IDDomainSpotter: compositional bias reveals domains in long disordered protein regions — insights from transcription factors. Protein Sci. 29, 169–183 (2020).
Lotthammer, J. M., Ginell, G. M., Griffith, D., Emenecker, R. J. & Holehouse, A. S. Direct prediction of intrinsically disordered protein conformational properties from sequence. Nat. Methods (in press).
Holehouse, A. S., Das, R. K., Ahad, J. N., Richardson, M. O. G. & Pappu, R. V. CIDER: resources to analyze sequence–ensemble relationships of intrinsically disordered proteins. Biophys. J. 112, 16–21 (2017).
Xue, B., Dunker, A. K. & Uversky, V. N. Orderly order in protein intrinsic disorder distribution: disorder in 3500 proteomes from viruses and the three domains of life. J. Biomol. Struct. Dyn. 30, 137–149 (2012).
Cohan, M. C. & Pappu, R. V. Making the case for disordered proteins and biomolecular condensates in bacteria. Trends Biochem. Sci. 45, 668–680 (2020).
Dyson, H. J. & Wright, P. E. Equilibrium NMR studies of unfolded and partially folded proteins. Nat. Struct. Biol. 5, 499–503 (1998). Along with Wright and Dyson (1999), this perspective article makes the case that an ensemble-centric biophysical lens is crucial for understanding disordered proteins.
Mittag, T. & Forman-Kay, J. D. Atomic-level characterization of disordered protein ensembles. Curr. Opin. Struct. Biol. 17, 3–14 (2007).
Henzler-Wildman, K. & Kern, D. Dynamic personalities of proteins. Nature 450, 964–972 (2007).
Babu, M. M., Kriwacki, R. W. & Pappu, R. V. Structural biology. Versatility from protein disorder. Science 337, 1460–1461 (2012).
Lazar, T. et al. PED in 2021: a major update of the protein ensemble database for intrinsically disordered proteins. Nucleic Acids Res. 49, D404–D411 (2021).
Das, R. K., Ruff, K. M. & Pappu, R. V. Relating sequence encoded information to form and function of intrinsically disordered proteins. Curr. Opin. Struct. Biol. 32, 102–112 (2015).
Mao, A. H., Lyle, N. & Pappu, R. V. Describing sequence–ensemble relationships for intrinsically disordered proteins. Biochem. J. 449, 307–318 (2013).
Crabtree, M. D. et al. Conserved helix-flanking prolines modulate intrinsically disordered protein: target affinity by altering the lifetime of the bound complex. Biochemistry 56, 2379–2384 (2017).
Wicky, B. I. M., Shammas, S. L. & Clarke, J. Affinity of IDPs to their targets is modulated by ion-specific changes in kinetics and residual structure. Proc. Natl Acad. Sci. USA 114, 9882–9887 (2017).
Dyla, M., González Foutel, N. S., Otzen, D. E. & Kjaergaard, M. The optimal docking strength for reversibly tethered kinases. Proc. Natl Acad. Sci. USA 119, e2203098119 (2022).
González-Foutel, N. S. et al. Conformational buffering underlies functional selection in intrinsically disordered protein regions. Nat. Struct. Mol. Biol. 29, 781–790 (2022). In this study, the authors present evidence that a viral IDR linker is conserved with respect to ensemble dimensions, despite large-scale sequence and length variation.
Harmon, T. S., Holehouse, A. S., Rosen, M. K. & Pappu, R. V. Intrinsically disordered linkers determine the interplay between phase separation and gelation in multivalent proteins. eLife 6, e30294 (2017).
Huang, Q., Li, M., Lai, L. & Liu, Z. Allostery of multidomain proteins with disordered linkers. Curr. Opin. Struct. Biol. 62, 175–182 (2020).
Bugge, K. et al. Interactions by disorder — a matter of context. Front. Mol. Biosci. 7, 110 (2020). This review synthesizes a core and emerging idea in the field of disordered proteins; that IDR function is strongly influenced by context.
Sturzenegger, F. et al. Transition path times of coupled folding and binding reveal the formation of an encounter complex. Nat. Commun. 9, 4708 (2018).
Holmstrom, E. D., Liu, Z., Nettels, D., Best, R. B. & Schuler, B. Disordered RNA chaperones can enhance nucleic acid folding via local charge screening. Nat. Commun. 10, 2453 (2019).
Cubuk, J. et al. The disordered N-terminal tail of SARS CoV-2 nucleocapsid protein forms a dynamic complex with RNA. Preprint at bioRxiv https://doi.org/10.1101/2023.02.10.527914 (2023).
Stuchell-Brereton, M. D. et al. Apolipoprotein E4 has extensive conformational heterogeneity in lipid-free and lipid-bound forms. Proc. Natl Acad. Sci. USA 120, e2215371120 (2023).
Moses, D., Ginell, G. M., Holehouse, A. S. & Sukenik, S. Intrinsically disordered regions are poised to act as sensors of cellular chemistry. Trends Biochem. Sci. https://doi.org/10.1016/j.tibs.2023.08.001 (2023).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Tunyasuvunakool, K. et al. Highly accurate protein structure prediction for the human proteome. Nature 596, 590–596 (2021).
Baek, M. et al. Accurate prediction of protein structures and interactions using a three-track neural network. Science 373, 871–876 (2021).
Ruff, K. M. & Pappu, R. V. AlphaFold and implications for intrinsically disordered proteins. J. Mol. Biol. 433, 167208 (2021).
Necci, M., Piovesan, D., CAID Predictors, DisProt Curators & Tosatto, S. C. E. Critical assessment of protein intrinsic disorder prediction. Nat. Methods 18, 472–481 (2021).
Conte, A. D. et al. Critical assessment of protein intrinsic disorder prediction (CAID) — results of round 2. Proteins https://doi.org/10.1002/prot.26582 (2023).
Brown, C. J., Johnson, A. K., Dunker, A. K. & Daughdrill, G. W. Evolution and disorder. Curr. Opin. Struct. Biol. 21, 441–446 (2011).
Brown, C. J., Johnson, A. K. & Daughdrill, G. W. Comparing models of evolution for ordered and disordered proteins. Mol. Biol. Evol. 27, 609–621 (2010).
Zarin, T. et al. Proteome-wide signatures of function in highly diverged intrinsically disordered regions. eLife 8, e46883 (2019). This study offers a systematic assessment of how conservation in IDRs could act at the level of sequence features instead of specific linear sequence.
Cohan, M. C., Shinn, M. K., Lalmansingh, J. M. & Pappu, R. V. Uncovering non-random binary patterns within sequences of intrinsically disordered proteins. J. Mol. Biol. 434, 167373 (2022).
Martin, E. W. et al. Valence and patterning of aromatic residues determine the phase behavior of prion-like domains. Science 367, 694–699 (2020). This study provides a detailed biophysical assessment of how sequence features and ensemble properties cooperate to determine the driving forces for phase separation in low-complexity disordered regions.
Beh, L. Y., Colwell, L. J. & Francis, N. J. A core subunit of Polycomb repressive complex 1 is broadly conserved in function but not primary sequence. Proc. Natl Acad. Sci. USA 109, E1063–E1071 (2012).
Zarin, T., Tsai, C. N., Nguyen Ba, A. N. & Moses, A. M. Selection maintains signaling function of a highly diverged intrinsically disordered region. Proc. Natl Acad. Sci. USA 114, E1450–E1459 (2017).
Tesei, G. et al. Conformational ensembles of the human intrinsically disordered proteome: bridging chain compaction with function and sequence conservation. Preprint at bioRxiv https://doi.org/10.1101/2023.05.08.539815 (2023).
Martin, E. W. et al. Sequence determinants of the conformational properties of an intrinsically disordered protein prior to and upon multisite phosphorylation. J. Am. Chem. Soc. 138, 15323–15335 (2016).
Müller-Späth, S. et al. Charge interactions can dominate the dimensions of intrinsically disordered proteins. Proc. Natl Acad. Sci. USA 107, 14609–14614 (2010). Along with Marsh and Forman-Kay (2010) and Mao et al. (2010), this paper offers direct evidence that net charge of IDRs influences global dimensions.
Marsh, J. A. & Forman-Kay, J. D. Sequence determinants of compaction in intrinsically disordered proteins. Biophys. J. 98, 2383–2390 (2010).
Mao, A. H., Crick, S. L., Vitalis, A., Chicoine, C. L. & Pappu, R. V. Net charge per residue modulates conformational ensembles of intrinsically disordered proteins. Proc. Natl Acad. Sci. USA 107, 8183–8188 (2010).
Das, R. K. & Pappu, R. V. Conformations of intrinsically disordered proteins are influenced by linear sequence distributions of oppositely charged residues. Proc. Natl Acad. Sci. USA 110, 13392–13397 (2013). This study is the first to systematically show that the patterning of charged residues in IDRs can be an important feature that influences ensemble behaviour.
Sawle, L. & Ghosh, K. A theoretical method to compute sequence dependent configurational properties in charged polymers and proteins. J. Chem. Phys. 143, 085101 (2015).
Kulkarni, P. et al. Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity. Proc. Natl Acad. Sci. USA 114, E2644–E2653 (2017).
Jin, F. & Gräter, F. How multisite phosphorylation impacts the conformations of intrinsically disordered proteins. PLoS Comput. Biol. 17, e1008939 (2021).
Sørensen, C. S. & Kjaergaard, M. Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics. Proc. Natl Acad. Sci. USA 116, 23124–23131 (2019).
Zeng, X., Ruff, K. M. & Pappu, R. V. Competing interactions give rise to two-state behavior and switch-like transitions in charge-rich intrinsically disordered proteins. Proc. Natl Acad. Sci. USA 119, e2200559119 (2022).
Portz, B. et al. Structural heterogeneity in the intrinsically disordered RNA polymerase II C-terminal domain. Nat. Commun. 8, 15231 (2017).
Bremer, A. et al. Deciphering how naturally occurring sequence features impact the phase behaviours of disordered prion-like domains. Nat. Chem. 14, 196–207 (2022).
Plevin, M. J., Bryce, D. L. & Boisbouvier, J. Direct detection of CH/pi interactions in proteins. Nat. Chem. 2, 466–471 (2010).
Crick, S. L., Jayaraman, M., Frieden, C., Wetzel, R. & Pappu, R. V. Fluorescence correlation spectroscopy shows that monomeric polyglutamine molecules form collapsed structures in aqueous solutions. Proc. Natl Acad. Sci. USA 103, 16764–16769 (2006).
Holehouse, A. S., Garai, K., Lyle, N., Vitalis, A. & Pappu, R. V. Quantitative assessments of the distinct contributions of polypeptide backbone amides versus side chain groups to chain expansion via chemical denaturation. J. Am. Chem. Soc. 137, 2984–2995 (2015).
Mukhopadhyay, S., Krishnan, R., Lemke, E. A., Lindquist, S. & Deniz, A. A. A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures. Proc. Natl Acad. Sci. USA 104, 2649–2654 (2007).
Theillet, F.-X. et al. The alphabet of intrinsic disorder: I. Act like a pro: on the abundance and roles of proline residues in intrinsically disordered proteins. Intrinsically Disord. Proteins 1, e24360 (2013).
Boze, H. et al. Proline-rich salivary proteins have extended conformations. Biophys. J. 99, 656–665 (2010).
Gibbs, E. B. et al. Phosphorylation induces sequence-specific conformational switches in the RNA polymerase II C-terminal domain. Nat. Commun. 8, 15233 (2017).
Rauscher, S., Baud, S., Miao, M., Keeley, F. W. & Pomès, R. Proline and glycine control protein self-organization into elastomeric or amyloid fibrils. Structure 14, 1667–1676 (2006).
Beveridge, R. et al. Ion mobility mass spectrometry uncovers the impact of the patterning of oppositely charged residues on the conformational distributions of intrinsically disordered proteins. J. Am. Chem. Soc. 141, 4908–4918 (2019).
Das, R. K., Huang, Y., Phillips, A. H., Kriwacki, R. W. & Pappu, R. V. Cryptic sequence features within the disordered protein p27Kip1 regulate cell cycle signaling. Proc. Natl Acad. Sci. USA 113, 5616–5621 (2016).
Sherry, K. P., Das, R. K., Pappu, R. V. & Barrick, D. Control of transcriptional activity by design of charge patterning in the intrinsically disordered RAM region of the Notch receptor. Proc. Natl Acad. Sci. USA 114, E9243–E9252 (2017).
Ginell, G. M., Flynn, A. J. & Holehouse, A. S. SHEPHARD: a modular and extensible software architecture for analyzing and annotating large protein datasets. Bioinformatics 39, btad488 (2023).
Iakoucheva, L. M. et al. The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Res. 32, 1037–1049 (2004).
Choy, M. S., Page, R. & Peti, W. Regulation of protein phosphatase 1 by intrinsically disordered proteins. Biochem. Soc. Trans. 40, 969–974 (2012).
Gomes, G.-N. W. et al. Conformational ensembles of an intrinsically disordered protein consistent with NMR, SAXS, and single-molecule FRET. J. Am. Chem. Soc. 142, 15697–15710 (2020).
Nash, P. et al. Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature 414, 514–521 (2001).
Mittag, T. et al. Dynamic equilibrium engagement of a polyvalent ligand with a single-site receptor. Proc. Natl Acad. Sci. USA 105, 17772–17777 (2008).
Borg, M. et al. Polyelectrostatic interactions of disordered ligands suggest a physical basis for ultrasensitivity. Proc. Natl Acad. Sci. USA 104, 9650–9655 (2007).
Ruff, K. M. Predicting conformational properties of intrinsically disordered proteins from sequence. Methods Mol. Biol. 2141, 347–389 (2020).
Zarin, T. et al. Identifying molecular features that are associated with biological function of intrinsically disordered protein regions. eLife 10, e60220 (2021).
Ginell, G. M. & Holehouse, A. S. Analyzing the sequences of intrinsically disordered regions with CIDER and localCIDER. in Intrinsically Disordered Proteins: Methods and Protocols Vol. 2141 (eds Kragelund, B. B. & Skriver, K.) 103–126 (Springer, 2020).
Ghosh, K., Huihui, J., Phillips, M. & Haider, A. Rules of physical mathematics govern intrinsically disordered proteins. Annu. Rev. Biophys. https://doi.org/10.1146/annurev-biophys-120221-095357 (2022).
Huihui, J. & Ghosh, K. Intra-chain interaction topology can identify functionally similar intrinsically disordered proteins. Biophys. J. https://doi.org/10.1016/j.bpj.2020.11.2282 (2021).
Huihui, J. & Ghosh, K. An analytical theory to describe sequence-specific inter-residue distance profiles for polyampholytes and intrinsically disordered proteins. J. Chem. Phys. 152, 161102 (2020).
Franzmann, T. M. et al. Phase separation of a yeast prion protein promotes cellular fitness. Science 359, eaao5654 (2018).
Yamazaki, H., Takagi, M., Kosako, H., Hirano, T. & Yoshimura, S. H. Cell cycle-specific phase separation regulated by protein charge blockiness. Nat. Cell Biol. 24, 625–632 (2022).
Lyons, H. et al. Functional partitioning of transcriptional regulators by patterned charge blocks. Cell 186, 327–345.e28 (2023). In this paper, the authors show that specific patterning of charged residues in IDRs enables specific molecular recruitment to transcriptional condensates.
Jankowski, M. S. et al. The formation of a fuzzy complex in the negative arm regulates the robustness of the circadian clock. Preprint at bioRxiv https://doi.org/10.1101/2022.01.04.474980 (2022).
Brendel, V. & Karlin, S. Association of charge clusters with functional domains of cellular transcription factors. Proc. Natl Acad. Sci. USA 86, 5698–5702 (1989).
Robustelli, P., Piana, S. & Shaw, D. E. Mechanism of coupled folding-upon-binding of an intrinsically disordered protein. J. Am. Chem. Soc. 142, 11092–11101 (2020).
Das, R. K., Crick, S. L. & Pappu, R. V. N-terminal segments modulate the α-helical propensities of the intrinsically disordered basic regions of bZIP proteins. J. Mol. Biol. 416, 287–299 (2012).
Milles, S. et al. An ultraweak interaction in the intrinsically disordered replication machinery is essential for measles virus function. Sci. Adv. 4, eaat7778 (2018).
Daughdrill, G. W. Disorder for dummies: functional mutagenesis of transient helical segments in disordered proteins. Methods Mol. Biol. 2141, 3–20 (2020).
Davey, N. E. The functional importance of structure in unstructured protein regions. Curr. Opin. Struct. Biol. 56, 155–163 (2019).
Zhu, J., Salvatella, X. & Robustelli, P. Small molecules targeting the disordered transactivation domain of the androgen receptor induce the formation of collapsed helical states. Nat. Commun. 13, 6390 (2022).
Chhabra, Y. et al. A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation. Oncogene 37, 489–501 (2018).
Loening, N. M., Saravanan, S., Jespersen, N. E., Jara, K. & Barbar, E. Interplay of disorder and sequence specificity in the formation of stable dynein–dynactin complexes. Biophys. J. 119, 950–965 (2020).
Clark, S. et al. Multivalency regulates activity in an intrinsically disordered transcription factor. eLife 7, e36258 (2018).
Burke, K. A., Janke, A. M., Rhine, C. L. & Fawzi, N. L. Residue-by-residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase II. Mol. Cell 60, 231–241 (2015).
Murthy, A. C. et al. Molecular interactions underlying liquid–liquid phase separation of the FUS low-complexity domain. Nat. Struct. Mol. Biol. 26, 637–648 (2019).
Murthy, A. C. et al. Molecular interactions contributing to FUS SYGQ LC-RGG phase separation and co-partitioning with RNA polymerase II heptads. Nat. Struct. Mol. Biol. 28, 923–935 (2021).
Yang, P. et al. G3BP1 is a tunable switch that triggers phase separation to assemble stress granules. Cell 181, 325–345.e28 (2020).
Guillén-Boixet, J. et al. RNA-induced conformational switching and clustering of G3BP drive stress granule assembly by condensation. Cell 181, 346–361.e17 (2020).
Warner, J. B. IV et al. Monomeric huntingtin exon 1 has similar overall structural features for wild-type and pathological polyglutamine lengths. J. Am. Chem. Soc. 139, 14456–14469 (2017).
Newcombe, E. A. et al. Tadpole-like conformations of huntingtin exon 1 are characterized by conformational heterogeneity that persists regardless of polyglutamine length. J. Mol. Biol. 430, 1442–1458 (2018).
Urbanek, A. et al. Flanking regions determine the structure of the poly-glutamine in huntingtin through mechanisms common among glutamine-rich human proteins. Structure 28, 733–746.e5 (2020).
Elena-Real, C. A. et al. The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity. Nat. Struct. Mol. Biol. 30, 309–320 (2023).
Moses, D. et al. Revealing the hidden sensitivity of intrinsically disordered proteins to their chemical environment. J. Phys. Chem. Lett. 11, 10131–10136 (2020). In this work, the authors combine experiment, simulation and theory to examine how IDRs show sequence-dependent sensitivity to changes in the solution context.
Sørensen, C. S. & Kjaergaard, M. Measuring effective concentrations enforced by intrinsically disordered linkers. Methods Mol. Biol. 2141, 505–518 (2020).
Mateos, B. et al. Hyperphosphorylation of human osteopontin and its impact on structural dynamics and molecular recognition. Biochemistry https://doi.org/10.1021/acs.biochem.1c00050 (2021).
Dignon, G. L., Zheng, W., Best, R. B., Kim, Y. C. & Mittal, J. Relation between single-molecule properties and phase behavior of intrinsically disordered proteins. Proc. Natl Acad. Sci. USA 115, 9929–9934 (2018).
Keul, N. D. et al. The entropic force generated by intrinsically disordered segments tunes protein function. Nature 563, 584–588 (2018).
Busch, D. J. et al. Intrinsically disordered proteins drive membrane curvature. Nat. Commun. 6, 7875 (2015).
Borcherds, W. et al. Optimal affinity enhancement by a conserved flexible linker controls p53 mimicry in MdmX. Biophys. J. 112, 2038–2042 (2017).
Kjaergaard, M. Estimation of effective concentrations enforced by complex linker architectures from conformational ensembles. Biochemistry 61, 171–182 (2022).
Martin, I. M. et al. Phosphorylation tunes elongation propensity and cohesiveness of INCENP’s intrinsically disordered region. J. Mol. Biol. 434, 167387 (2022).
Sherry, K. P., Johnson, S. E., Hatem, C. L., Majumdar, A. & Barrick, D. Effects of linker length and transient secondary structure elements in the intrinsically disordered notch RAM region on notch signaling. J. Mol. Biol. 427, 3587–3597 (2015).
Motlagh, H. N., Wrabl, J. O., Li, J. & Hilser, V. J. The ensemble nature of allostery. Nature 508, 331–339 (2014).
Li, M., Cao, H., Lai, L. & Liu, Z. Disordered linkers in multidomain allosteric proteins: entropic effect to favor the open state or enhanced local concentration to favor the closed state? Protein Sci. 27, 1600–1610 (2018).
Huang, W. Y. C., Ditlev, J. A., Chiang, H.-K., Rosen, M. K. & Groves, J. T. Allosteric modulation of Grb2 recruitment to the intrinsically disordered scaffold protein, LAT, by remote site phosphorylation. J. Am. Chem. Soc. 139, 18009–18015 (2017).
Seiffert, P. et al. Orchestration of signaling by structural disorder in class 1 cytokine receptors. Cell Commun. Signal. 18, 132 (2020).
Yu, F. & Sukenik, S. Structural preferences shape the entropic force of disordered protein ensembles. J. Phys. Chem. B 127, 4235–4244 (2023).
Zeno, W. F. et al. Synergy between intrinsically disordered domains and structured proteins amplifies membrane curvature sensing. Nat. Commun. 9, 4152 (2018).
Zeno, W. F. et al. Molecular mechanisms of membrane curvature sensing by a disordered protein. J. Am. Chem. Soc. 141, 10361–10371 (2019).
Halladin, D. K. et al. Entropy-driven translocation of disordered proteins through the Gram-positive bacterial cell wall. Nat. Microbiol. 6, 1055–1065 (2021).
Davey, N. E. et al. Attributes of short linear motifs. Mol. Biosyst. 8, 268–281 (2012).
Holehouse, A. S. IDPs and IDRs in biomolecular condensates. in Intrinsically Disordered Proteins (ed. Salvi, N.) Ch. 7, 209–255 (Academic Press, 2019).
Posey, A. E., Holehouse, A. S. & Pappu, R. V. Phase separation of intrinsically disordered proteins. in Methods in Enzymology Vol. 611 (ed. Rhoades, E.) Ch. 1, 1–30 (Academic Press, 2018).
Brangwynne, C. P., Tompa, P. & Pappu, R. V. Polymer physics of intracellular phase transitions. Nat. Phys. 11, 899–904 (2015).
Duchesne, L. et al. Transport of fibroblast growth factor 2 in the pericellular matrix is controlled by the spatial distribution of its binding sites in heparan sulfate. PLoS Biol. 10, e1001361 (2012).
Kleinschmit, A. et al. Drosophila heparan sulfate 6-O endosulfatase regulates wingless morphogen gradient formation. Dev. Biol. 345, 204–214 (2010).
Yan, D. & Lin, X. Shaping morphogen gradients by proteoglycans. Cold Spring Harb. Perspect. Biol. 1, a002493 (2009).
Triandafillou, C. G., Katanski, C. D., Dinner, A. R. & Drummond, D. A. Transient intracellular acidification regulates the core transcriptional heat shock response. eLife 9, e54880 (2020).
Gutierrez, J. I. et al. SWI/SNF senses carbon starvation with a pH-sensitive low-complexity sequence. eLife 11, e70344 (2022).
Soranno, A. et al. Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments. Proc. Natl Acad. Sci. USA 111, 4874–4879 (2014).
Delarue, M. et al. mTORC1 controls phase separation and the biophysical properties of the cytoplasm by tuning crowding. Cell 174, 338–349.e20 (2018).
Battaglia, M., Olvera-Carrillo, Y., Garciarrubio, A., Campos, F. & Covarrubias, A. A. The enigmatic LEA proteins and other hydrophilins. Plant Physiol. 148, 6–24 (2008).
Boothby, T. C. & Pielak, G. J. Intrinsically disordered proteins and desiccation tolerance: elucidating functional and mechanistic underpinnings of anhydrobiosis. Bioessays 39, 1700119 (2017).
Boothby, T. C. et al. Tardigrades use intrinsically disordered proteins to survive desiccation. Mol. Cell 65, 975–984.e5 (2017).
Wuttke, R. et al. Temperature-dependent solvation modulates the dimensions of disordered proteins. Proc. Natl Acad. Sci. USA 111, 5213–5218 (2014).
Quiroz, F. G. & Chilkoti, A. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers. Nat. Mater. 14, 1164–1171 (2015).
Kjaergaard, M. et al. Temperature-dependent structural changes in intrinsically disordered proteins: formation of alpha-helices or loss of polyproline II? Protein Sci. 19, 1555–1564 (2010).
Jung, J.-H. et al. A prion-like domain in ELF3 functions as a thermosensor in Arabidopsis. Nature 585, 256–260 (2020).
Zhu, P., Lister, C. & Dean, C. Cold-induced Arabidopsis FRIGIDA nuclear condensates for FLC repression. Nature 599, 657–661 (2021).
Cuylen-Haering, S. et al. Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly. Nature 587, 285–290 (2020).
Kumar, S. & Hoh, J. H. Modulation of repulsive forces between neurofilaments by sidearm phosphorylation. Biochem. Biophys. Res. Commun. 324, 489–496 (2004).
Doncic, A. et al. Compartmentalization of a bistable switch enables memory to cross a feedback-driven transition. Cell 160, 1182–1195 (2015).
Kõivomägi, M. et al. Multisite phosphorylation networks as signal processors for Cdk1. Nat. Struct. Mol. Biol. 20, 1415–1424 (2013).
Zhou, M., Kim, J. K., Eng, G. W. L., Forger, D. B. & Virshup, D. M. A Period2 phosphoswitch regulates and temperature compensates circadian period. Mol. Cell 60, 77–88 (2015).
Bah, A. et al. Folding of an intrinsically disordered protein by phosphorylation as a regulatory switch. Nature 519, 106–109 (2015). Here the authors show that multisite phosphorylation of a disordered region can mediate intramolecular folding into a stable 3D structure, offering an example of phospho-conditional folding.
Mittal, A., Holehouse, A. S., Cohan, M. C. & Pappu, R. V. Sequence-to-conformation relationships of disordered regions tethered to folded domains of proteins. J. Mol. Biol. 430, 2403–2421 (2018).
Taneja, I. & Holehouse, A. S. Folded domain charge properties influence the conformational behavior of disordered tails. Curr. Res. Struct. Biol. 3, 216–228 (2021).
Zheng, T., Galagedera, S. K. K. & Castañeda, C. A. Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation. Protein Sci. 30, 1467-1481 (2021).
Martin, E. W. et al. Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation. Nucleic Acids Res. 49, 2931–2945 (2021).
Bjarnason, S. et al. DNA binding redistributes activation domain ensemble and accessibility in pioneer factor Sox2. Preprint at bioRxiv https://doi.org/10.1101/2023.06.16.545083 (2023).
Farr, S. E., Woods, E. J., Joseph, J. A., Garaizar, A. & Collepardo-Guevara, R. Nucleosome plasticity is a critical element of chromatin liquid–liquid phase separation and multivalent nucleosome interactions. Nat. Commun. 12, 2883 (2021).
Heidarsson, P. O. et al. Release of linker histone from the nucleosome driven by polyelectrolyte competition with a disordered protein. Nat. Chem. 14, 224–231 (2022).
Chen, Q., Yang, R., Korolev, N., Liu, C. F. & Nordenskiöld, L. Regulation of nucleosome stacking and chromatin compaction by the histone H4 N-terminal tail–H2A acidic patch interaction. J. Mol. Biol. 429, 2075–2092 (2017).
Staby, L. et al. Flanking disorder of the folded αα-hub domain from radical induced cell death1 affects transcription factor binding by ensemble redistribution. J. Mol. Biol. 433, 167320 (2021).
Staby, L. et al. Disorder in a two-domain neuronal Ca2+-binding protein regulates domain stability and dynamics using ligand mimicry. Cell. Mol. Life Sci. 78, 2263–2278 (2021).
Corless, E. I. et al. The flexible N-terminus of BchL autoinhibits activity through interaction with its [4Fe-4S] cluster and released upon ATP binding. J. Biol. Chem. 296, 100107 (2021).
Alston, J. J., Soranno, A. & Holehouse, A. S. Conserved molecular recognition by an intrinsically disordered region in the absence of sequence conservation. Preprint at bioRxiv https://doi.org/10.1101/2023.08.06.552128 (2023).
Hendus-Altenburger, R. et al. A phosphorylation-motif for tuneable helix stabilisation in intrinsically disordered proteins — lessons from the sodium proton exchanger 1 (NHE1). Cell Signal. 37, 40–51 (2017).
Hendus-Altenburger, R. et al. The human Na(+)/H(+) exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2. BMC Biol. 14, 31 (2016).
Brown, C. J. et al. Evolutionary rate heterogeneity in proteins with long disordered regions. J. Mol. Evol. 55, 104–110 (2002).
Langstein-Skora, I. et al. Sequence- and chemical specificity define the functional landscape of intrinsically disordered regions. Preprint at bioRxiv https://doi.org/10.1101/2022.02.10.480018 (2022).
Davey, N. E., Cyert, M. S. & Moses, A. M. Short linear motifs — ex nihilo evolution of protein regulation. Cell Commun. Signal. 13, 43 (2015).
Sangster, A. G., Zarin, T. & Moses, A. M. Evolution of short linear motifs and disordered proteins topic: yeast as model system to study evolution. Curr. Opin. Genet. Dev. 76, 101964 (2022).
Garboczi, D. N. et al. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature 384, 134–141 (1996).
Rebek, J. Jr. Model studies in molecular recognition. Science 235, 1478–1484 (1987).
Brooijmans, N. & Kuntz, I. D. Molecular recognition and docking algorithms. Annu. Rev. Biophys. Biomol. Struct. 32, 335–373 (2003).
Rogers, J. M., Wong, C. T. & Clarke, J. Coupled folding and binding of the disordered protein PUMA does not require particular residual structure. J. Am. Chem. Soc. 136, 5197–5200 (2014).
Chen, T., Song, J. & Chan, H. S. Theoretical perspectives on nonnative interactions and intrinsic disorder in protein folding and binding. Curr. Opin. Struct. Biol. 30, 32–42 (2014).
Ivarsson, Y. & Jemth, P. Affinity and specificity of motif-based protein–protein interactions. Curr. Opin. Struct. Biol. 54, 26–33 (2019).
Staller, M. V. et al. Directed mutational scanning reveals a balance between acidic and hydrophobic residues in strong human activation domains. Cell Syst. 13, 334–345.e5 (2022).
Berlow, R. B., Dyson, H. J. & Wright, P. E. Hypersensitive termination of the hypoxic response by a disordered protein switch. Nature 543, 447–451 (2017). In this study, the authors reveal how dynamic intermolecular competition coupled with disorder-driven allosteric changes can lead to complex regulatory behaviour in IDR binding.
Olsen, J. G., Teilum, K. & Kragelund, B. B. Behaviour of intrinsically disordered proteins in protein–protein complexes with an emphasis on fuzziness. Cell. Mol. Life Sci. 74, 3175–3183 (2017).
Tompa, P. & Fuxreiter, M. Fuzzy complexes: polymorphism and structural disorder in protein–protein interactions. Trends Biochem. Sci. 33, 2–8 (2008).
Eick, D. & Geyer, M. The RNA polymerase II carboxy-terminal domain (CTD) code. Chem. Rev. 113, 8456–8490 (2013).
Prestel, A. et al. The PCNA interaction motifs revisited: thinking outside the PIP-box. Cell. Mol. Life Sci. 76, 4923–4943 (2019).
Follis, A. V. et al. Regulation of apoptosis by an intrinsically disordered region of Bcl-xL. Nat. Chem. Biol. 14, 458–465 (2018).
He, F. et al. Interaction between p53 N terminus and core domain regulates specific and nonspecific DNA binding. Proc. Natl Acad. Sci. USA 116, 8859–8868 (2019).
Krois, A. S., Dyson, H. J. & Wright, P. E. Long-range regulation of p53 DNA binding by its intrinsically disordered N-terminal transactivation domain. Proc. Natl Acad. Sci. USA 115, E11302–E11310 (2018).
Jenuwein, T. & Allis, C. D. Translating the histone code. Science 293, 1074–1080 (2001).
Phatnani, H. P. & Greenleaf, A. L. Phosphorylation and functions of the RNA polymerase II CTD. Genes Dev. 20, 2922–2936 (2006).
Wright, P. E. & Dyson, H. J. Linking folding and binding. Curr. Opin. Struct. Biol. 19, 31–38 (2009).
Sugase, K., Dyson, H. J. & Wright, P. E. Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447, 1021–1025 (2007).
Rogers, J. M., Steward, A. & Clarke, J. Folding and binding of an intrinsically disordered protein: fast, but not ‘diffusion-limited’. J. Am. Chem. Soc. 135, 1415–1422 (2013).
Gianni, S., Morrone, A., Giri, R. & Brunori, M. A folding-after-binding mechanism describes the recognition between the transactivation domain of c-Myb and the KIX domain of the CREB-binding protein. Biochem. Biophys. Res. Commun. 428, 205–209 (2012).
Dogan, J., Schmidt, T., Mu, X., Engström, Å. & Jemth, P. Fast association and slow transitions in the interaction between two intrinsically disordered protein domains. J. Biol. Chem. 287, 34316–34324 (2012).
Teilum, K., Olsen, J. G. & Kragelund, B. B. Globular and disordered — the non-identical twins in protein–protein interactions. Front. Mol. Biosci. 2, 40 (2015).
Lazar, T., Tantos, A., Tompa, P. & Schad, E. Intrinsic protein disorder uncouples affinity from binding specificity. Protein Sci. 31, e4455 (2022).
Skriver, K., Theisen, F. F. & Kragelund, B. B. Conformational entropy in molecular recognition of intrinsically disordered proteins. Curr. Opin. Struct. Biol. 83, 102697 (2023).
O’Shea, C. et al. Structures and short linear motif of disordered transcription factor regions provide clues to the interactome of the cellular hub protein radical-induced cell death1. J. Biol. Chem. 292, 512–527 (2017).
Theisen, F. F. et al. αα-Hub coregulator structure and flexibility determine transcription factor binding and selection in regulatory interactomes. J. Biol. Chem. 298, 101963 (2022).
Arai, M., Sugase, K., Dyson, H. J. & Wright, P. E. Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and folding. Proc. Natl Acad. Sci. USA 112, 9614–9619 (2015).
Shammas, S. L., Crabtree, M. D., Dahal, L., Wicky, B. I. M. & Clarke, J. Insights into coupled folding and binding mechanisms from kinetic studies. J. Biol. Chem. 291, 6689–6695 (2016).
Gianni, S., Dogan, J. & Jemth, P. Distinguishing induced fit from conformational selection. Biophys. Chem. 189, 33–39 (2014).
Dogan, J. & Jemth, P. Only kinetics can prove conformational selection. Biophys. J. 107, 1997–1998 (2014).
Hammes, G. G., Chang, Y.-C. & Oas, T. G. Conformational selection or induced fit: a flux description of reaction mechanism. Proc. Natl Acad. Sci. USA 106, 13737–13741 (2009).
Dyson, H. J. & Wright, P. E. Coupling of folding and binding for unstructured proteins. Curr. Opin. Struct. Biol. 12, 54–60 (2002).
Spolar, R. S. & Record, M. T. Jr. Coupling of local folding to site-specific binding of proteins to DNA. Science 263, 777–784 (1994).
Suarez, I. P., Burdisso, P., Benoit, M. P. M. H., Boisbouvier, J. & Rasia, R. M. Induced folding in RNA recognition by Arabidopsis thaliana DCL1. Nucleic Acids Res. 43, 6607–6619 (2015).
Vise, P. D., Baral, B., Latos, A. J. & Daughdrill, G. W. NMR chemical shift and relaxation measurements provide evidence for the coupled folding and binding of the p53 transactivation domain. Nucleic Acids Res. 33, 2061–2077 (2005).
Lee, C. W., Martinez-Yamout, M. A., Dyson, H. J. & Wright, P. E. Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein. Biochemistry 49, 9964–9971 (2010).
Elkjær, S. et al. Evolutionary fine-tuning of residual helix structure in disordered proteins manifests in complex structure and lifetime. Commun. Biol. 6, 63 (2023).
Crabtree, M. D., Mendonça, C. A. T. F., Bubb, Q. R. & Clarke, J. Folding and binding pathways of BH3-only proteins are encoded within their intrinsically disordered sequence, not templated by partner proteins. J. Biol. Chem. 293, 9718–9723 (2018).
Toto, A. et al. Molecular recognition by templated folding of an intrinsically disordered protein. Sci. Rep. 6, 21994 (2016).
Longhi, S. et al. The C-terminal domain of the measles virus nucleoprotein is intrinsically disordered and folds upon binding to the C-terminal moiety of the phosphoprotein. J. Biol. Chem. 278, 18638–18648 (2003).
Demarest, S. J. et al. Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators. Nature 415, 549–553 (2002).
Qin, B. Y. et al. Crystal structure of IRF-3 in complex with CBP. Structure 13, 1269–1277 (2005).
Karlsson, E. et al. Disordered regions flanking the binding interface modulate affinity between CBP and NCOA. J. Mol. Biol. 434, 167643 (2022).
Jemth, P. et al. Structure and dynamics conspire in the evolution of affinity between intrinsically disordered proteins. Sci. Adv. 4, eaau4130 (2018).
Fuxreiter, M. Fold or not to fold upon binding — does it really matter? Curr. Opin. Struct. Biol. 54, 19–25 (2019).
Sharma, R., Raduly, Z., Miskei, M. & Fuxreiter, M. Fuzzy complexes: specific binding without complete folding. FEBS Lett. 589, 2533–2542 (2015).
Jiang, Y., Rossi, P. & Kalodimos, C. G. Structural basis for client recognition and activity of Hsp40 chaperones. Science 365, 1313–1319 (2019).
Shi, Y. et al. Structure-based classification of tauopathies. Nature 598, 359–363 (2021).
Yang, Y. et al. Cryo-EM structures of amyloid-β 42 filaments from human brains. Science 375, 167–172 (2022).
Yang, Y. et al. Structures of α-synuclein filaments from human brains with Lewy pathology. Nature 610, 791–795 (2022).
Berlow, R. B., Dyson, H. J. & Wright, P. E. Multivalency enables unidirectional switch-like competition between intrinsically disordered proteins. Proc. Natl Acad. Sci. USA 119, e2117338119 (2022).
Tuttle, L. M. et al. Gcn4-mediator specificity is mediated by a large and dynamic fuzzy protein–protein complex. Cell Rep. 22, 3251–3264 (2018). In this study, the authors provide biophysical characterization of fuzzy transcription factor–co-activator binding.
Brzovic, P. S. et al. The acidic transcription activator Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex. Mol. Cell 44, 942–953 (2011).
Staller, M. V. et al. A high-throughput mutational scan of an intrinsically disordered acidic transcriptional activation domain. Cell Syst. 6, 444–455.e6 (2018).
Sanborn, A. L. et al. Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to mediator. eLife 10, e68068 (2021).
Görlich, D., Prehn, S., Laskey, R. A. & Hartmann, E. Isolation of a protein that is essential for the first step of nuclear protein import. Cell 79, 767–778 (1994).
Görlich, D. & Kutay, U. Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607–660 (1999).
Milles, S. et al. Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors. Cell 163, 734–745 (2015).
Frey, S. & Görlich, D. A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes. Cell 130, 512–523 (2007). This paper provides the first dissection of the sequence rules that determine what would now be referred to as biomolecular condensates, illustrating how IDRs can self-assemble into materials with biological activity.
Frey, S., Richter, R. P. & Görlich, D. FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. Science 314, 815–817 (2006).
Schmidt, H. B. & Görlich, D. Nup98 FG domains from diverse species spontaneously phase-separate into particles with nuclear pore-like permselectivity. eLife 4, e04251 (2015).
Buholzer, K. J. et al. Multilayered allosteric modulation of coupled folding and binding by phosphorylation, peptidyl-prolyl cis/trans isomerization, and diversity of interaction partners. J. Chem. Phys. 157, 235102 (2022).
Staller, M. V. Transcription factors perform a 2-step search of the nucleus. Genetics 222, iyac111 (2002).
Schuler, B. et al. Binding without folding — the biomolecular function of disordered polyelectrolyte complexes. Curr. Opin. Struct. Biol. 60, 66–76 (2019).
Borgia, A. et al. Extreme disorder in an ultrahigh-affinity protein complex. Nature 555, 61–66 (2018). This paper provides conclusive evidence that disordered proteins can form high-affinity biomolecular complexes that remain dynamic without the acquisition of any secondary or tertiary structure or persistent contacts.
Sottini, A. et al. Polyelectrolyte interactions enable rapid association and dissociation in high-affinity disordered protein complexes. Nat. Commun. 11, 5736 (2020).
Galvanetto, N. et al. Extreme dynamics in a biomolecular condensate. Nature 619, 876–883 (2023). In this work, the authors combine single-molecule studies and simulations to show that intra-condensate dynamics in IDR condensates can remain fast, despite macroscopic condensate viscosity being high.
Yu, M. et al. Visualizing the disordered nuclear transport machinery in situ. Nature 617, 162–169 (2023). In this work, the authors determine the conformational behaviour of phenylalanine-glycine-repeat IDRs inside the nuclear pore complex.
Frey, S. et al. Surface properties determining passage rates of proteins through nuclear pores. Cell 174, 202–217.e9 (2018).
Schneider, R., Blackledge, M. & Jensen, M. R. Elucidating binding mechanisms and dynamics of intrinsically disordered protein complexes using NMR spectroscopy. Curr. Opin. Struct. Biol. 54, 10–18 (2019).
Teilum, K., Olsen, J. G. & Kragelund, B. B. On the specificity of protein–protein interactions in the context of disorder. Biochem. J. 478, 2035–2050 (2021).
Eaton, B. E., Gold, L. & Zichi, D. A. Let’s get specific: the relationship between specificity and affinity. Chem. Biol. 2, 633–638 (1995).
Kumar, M. et al. ELM — the eukaryotic linear motif resource in 2020. Nucleic Acids Res. 48, D296–D306 (2019).
Tompa, P., Davey, N. E., Gibson, T. J. & Babu, M. M. A million peptide motifs for the molecular biologist. Mol. Cell 55, 161–169 (2014). This perspective article provides a clear assessment of the importance that SLiMs endow upon IDRs and their prevalence across eukaryotic proteomes.
Bruning, J. B. & Shamoo, Y. Structural and thermodynamic analysis of human PCNA with peptides derived from DNA polymerase-delta p66 subunit and flap endonuclease-1. Structure 12, 2209–2219 (2004).
Dreier, J. E. et al. A context-dependent and disordered ubiquitin-binding motif. Cell. Mol. Life Sci. 79, 484 (2022).
Lee, B. J. et al. Rules for nuclear localization sequence recognition by karyopherin β2. Cell 126, 543–558 (2006).
Oldfield, C. J. et al. Flexible nets: disorder and induced fit in the associations of p53 and 14-3-3 with their partners. BMC Genomics 9, S1 (2008).
Davey, N. E., Simonetti, L. & Ivarsson, Y. The next wave of interactomics: mapping the SLiM-based interactions of the intrinsically disordered proteome. Curr. Opin. Struct. Biol. 80, 102593 (2023).
Hadži, S., Loris, R. & Lah, J. The sequence–ensemble relationship in fuzzy protein complexes. Proc. Natl Acad. Sci. USA 118, e2020562118 (2021).
Nguyen, H. Q. et al. Quantitative mapping of protein–peptide affinity landscapes using spectrally encoded beads. eLife 8, e40499 (2019).
Mihalič, F. et al. Evolution of affinity between p53 transactivation domain and MDM2 across the animal kingdom demonstrates high plasticity of motif-mediated interactions. Protein Sci. 32, e4684 (2023).
Kumar, M. et al. The eukaryotic linear motif resource: 2022 release. Nucleic Acids Res. 50, D497–D508 (2022).
Davey, N. E., Shields, D. C. & Edwards, R. J. Masking residues using context-specific evolutionary conservation significantly improves short linear motif discovery. Bioinformatics 25, 443–450 (2009).
Chhabra, Y. et al. Tyrosine kinases compete for growth hormone receptor binding and regulate receptor mobility and degradation. Cell Rep. 42, 112490 (2023).
Raj, N. & Attardi, L. D. The transactivation domains of the p53 protein. Cold Spring Harb. Perspect. Med. 7, a026047 (2017).
Bochkareva, E. et al. Single-stranded DNA mimicry in the p53 transactivation domain interaction with replication protein A. Proc. Natl Acad. Sci. USA 102, 15412–15417 (2005).
Di Lello, P. et al. Structure of the Tfb1/p53 complex: insights into the interaction between the p62/Tfb1 subunit of TFIIH and the activation domain of p53. Mol. Cell 22, 731–740 (2006).
Miller Jenkins, L. M. et al. Characterization of the p300 Taz2–p53 TAD2 complex and comparison with the p300 Taz2–p53 TAD1 complex. Biochemistry 54, 2001–2010 (2015).
Rowell, J. P., Simpson, K. L., Stott, K., Watson, M. & Thomas, J. O. HMGB1-facilitated p53 DNA binding occurs via HMG-box/p53 transactivation domain interaction, regulated by the acidic tail. Structure 20, 2014–2024 (2012).
Lee, M.-S. et al. Solution structure of MUL1-RING domain and its interaction with p53 transactivation domain. Biochem. Biophys. Res. Commun. 516, 533–539 (2019).
Mészáros, B., Kumar, M., Gibson, T. J., Uyar, B. & Dosztányi, Z. Degrons in cancer. Sci. Signal. 10, eaak9982 (2017).
Provost, E. et al. Functional correlates of mutation of the Asp32 and Gly34 residues of beta-catenin. Oncogene 24, 2667–2676 (2005).
Olsen, J. G. et al. Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs. Preprint at bioRxiv https://doi.org/10.1101/2023.05.11.540346 (2023).
Meyer, K. et al. Mutations in disordered regions can cause disease by creating dileucine motifs. Cell 175, 239–253.e17 (2018).
Kliche, J. et al. Large-scale phosphomimetic screening identifies phospho-modulated motif-based protein interactions. Mol. Syst. Biol. 19, e11164 (2023).
Kassa, E. et al. Evaluation of affinity-purification coupled to mass spectrometry approaches for capture of short linear motif-based interactions. Anal. Biochem. 663, 115017 (2023).
Mihalič, F. et al. Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs. Nat. Commun. 14, 2409 (2023). In this study, a high-throughput analysis identified 1,712 SLiM-based virus–host interactions, in which viruses engage in molecular mimicry to subvert host programmes.
Stein, A. & Aloy, P. Contextual specificity in peptide-mediated protein interactions. PLoS ONE 3, e2524 (2008).
Karlsson, E., Ottoson, C., Ye, W., Andersson, E. & Jemth, P. Intrinsically disordered flanking regions increase the affinity of a transcriptional coactivator interaction across vertebrates. Biochemistry https://doi.org/10.1021/acs.biochem.3c00285 (2023).
Siligardi, G. et al. The SH3 domain of HS1 protein recognizes lysine-rich polyproline motifs. Amino Acids 42, 1361–1370 (2012).
Palopoli, N., González Foutel, N. S., Gibson, T. J. & Chemes, L. B. Short linear motif core and flanking regions modulate retinoblastoma protein binding affinity and specificity. Protein Eng. Des. Sel. 31, 69–77 (2018).
Brodsky, S. et al. Intrinsically disordered regions direct transcription factor in vivo binding specificity. Mol. Cell 79, 459–471.e4 (2020).
Strzalka, W., Oyama, T., Tori, K. & Morikawa, K. Crystal structures of the Arabidopsis thaliana proliferating cell nuclear antigen 1 and 2 proteins complexed with the human p21 C-terminal segment. Protein Sci. 18, 1072–1080 (2009).
Berlow, R. B., Martinez-Yamout, M. A., Dyson, H. J. & Wright, P. E. Role of backbone dynamics in modulating the interactions of disordered ligands with the TAZ1 domain of the CREB-binding protein. Biochemistry 58, 1354–1362 (2019).
Bhattacharyya, R. P. et al. The Ste5 scaffold allosterically modulates signaling output of the yeast mating pathway. Science 311, 822–826 (2006).
Mihalic, F. et al. Conservation of affinity rather than sequence underlies a dynamic evolution of the motif-mediated p53/MDM2 interaction in teleosts. Preprint at bioRxiv https://doi.org/10.1101/2023.08.24.554616 (2023).
Benz, C. et al. Proteome-scale mapping of binding sites in the unstructured regions of the human proteome. Mol. Syst. Biol. 18, e10584 (2022).
Jephthah, S., Staby, L., Kragelund, B. B. & Skepö, M. Temperature dependence of intrinsically disordered proteins in simulations: what are we missing? J. Chem. Theory Comput. 15, 2672–2683 (2019).
Cuevas-Velazquez, C. L. et al. Intrinsically disordered protein biosensor tracks the physical–chemical effects of osmotic stress on cells. Nat. Commun. 12, 5438 (2021).
Moses, D. et al. Structural biases in disordered proteins are prevalent in the cell. Nat. Struct. Mol. Biol. https://doi.org/10.1038/s41594-023-01148-8 (2023). In this work, the authors use in cell ensemble FRET to show that sequence-encoded conformational biases seen in vitro persist in the cellular environment and that changes to that environment can alter ensemble properties.
Klein, P., Pawson, T. & Tyers, M. Mathematical modeling suggests cooperative interactions between a disordered polyvalent ligand and a single receptor site. Curr. Biol. 13, 1669–1678 (2003).
Sparks, S., Hayama, R., Rout, M. P. & Cowburn, D. Analysis of multivalent IDP interactions: stoichiometry, affinity, and local concentration effect measurements. Methods Mol. Biol. 2141, 463–475 (2020).
Rogers, J. M. et al. Interplay between partner and ligand facilitates the folding and binding of an intrinsically disordered protein. Proc. Natl Acad. Sci. USA 111, 15420–15425 (2014).
Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).
Cho, W.-K. et al. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science 361, 412–415 (2018).
Chong, S. et al. Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science 361, eaar2555 (2018).
Brangwynne, C. P. et al. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729–1732 (2009).
Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science 357, eaaf4382 (2017).
Choi, J.-M., Holehouse, A. S. & Pappu, R. V. Physical principles underlying the complex biology of intracellular phase transitions. Annu. Rev. Biophys. 49, 107–133 (2020).
Dignon, G. L., Best, R. B. & Mittal, J. Biomolecular phase separation: from molecular driving forces to macroscopic properties. Annu. Rev. Phys. Chem. 71, 53–75 (2020).
Berry, J., Brangwynne, C. P. & Haataja, M. Physical principles of intracellular organization via active and passive phase transitions. Rep. Prog. Phys. 81, 046601 (2018).
Mittag, T. & Pappu, R. V. A conceptual framework for understanding phase separation and addressing open questions and challenges. Mol. Cell 82, 2201–2214 (2022).
Pappu, R. V., Cohen, S. R., Dar, F., Farag, M. & Kar, M. Phase transitions of associative biomacromolecules. Chem. Rev. 123, 8945–8987 (2023).
Boeynaems, S. et al. Protein phase separation: a new phase in cell biology. Trends Cell Biol. 28, 420–435 (2018).
Lyon, A. S., Peeples, W. B. & Rosen, M. K. A framework for understanding the functions of biomolecular condensates across scales. Nat. Rev. Mol. Cell Biol. 22, 1–21 (2020).
Martin, E. W. & Holehouse, A. S. Intrinsically disordered protein regions and phase separation: sequence determinants of assembly or lack thereof. Emerg. Top. Life Sci. 4, 307–329 (2020).
Li, P. et al. Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336–340 (2012).
Wang, J. et al. A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. Cell 174, 688–699.e16 (2018).
Choi, J.-M., Dar, F. & Pappu, R. V. LASSI: a lattice model for simulating phase transitions of multivalent proteins. PLoS Comput. Biol. 15, e1007028 (2019).
Choi, J.-M., Hyman, A. A. & Pappu, R. V. Generalized models for bond percolation transitions of associative polymers. Phys. Rev. E 102, 042403 (2020).
Ginell, G. M. & Holehouse, A. S. An introduction to the stickers-and-spacers framework as applied to biomolecular condensates. Methods Mol. Biol. 2563, 95–116 (2023).
Cates, M. E. & Witten, T. A. Chain conformation and solubility of associating polymers. Macromolecules 19, 732–739 (1986).
Semenov, A. N. & Rubinstein, M. Thermoreversible gelation in solutions of associative polymers. 1. Statics. Macromolecules 31, 1373–1385 (1998).
Rubinstein, M. & Semenov, A. N. Thermoreversible gelation in solutions of associating polymers. 2. Linear dynamics. Macromolecules 31, 1386–1397 (1998).
Rekhi, S. et al. Expanding the molecular language of protein liquid–liquid phase separation. Preprint at bioRxiv https://doi.org/10.1101/2023.03.02.530853 (2023).
Holehouse, A. S., Ginell, G. M., Griffith, D. & Böke, E. Clustering of aromatic residues in prion-like domains can tune the formation, state, and organization of biomolecular condensates. Biochemistry 60, 3566–3581 (2021).
Ruff, K. M. et al. Sequence grammar underlying the unfolding and phase separation of globular proteins. Mol. Cell 82, 3193–3208.e8 (2022).
Yang, Y., Jones, H. B., Dao, T. P. & Castañeda, C. A. Single amino acid substitutions in stickers, but not spacers, substantially alter UBQLN2 phase transitions and dense phase material properties. J. Phys. Chem. B 123, 3618–3629 (2019).
Farag, M. et al. Condensates formed by prion-like low-complexity domains have small-world network structures and interfaces defined by expanded conformations. Nat. Commun. 13, 7722 (2022).
Abbas, M., Lipiński, W. P., Nakashima, K. K., Huck, W. T. S. & Spruijt, E. A short peptide synthon for liquid–liquid phase separation. Nat. Chem. 13, 1046–1054 (2021).
Ruff, K. M., Dar, F. & Pappu, R. V. Ligand effects on phase separation of multivalent macromolecules. Proc. Natl Acad. Sci. USA 118, e2017184118 (2021).
Lin, Y.-H. & Chan, H. S. Phase separation and single-chain compactness of charged disordered proteins are strongly correlated. Biophys. J. 112, 2043–2046 (2017).
Banani, S. F. et al. Compositional control of phase-separated cell bodies. Cell 166, 651–663 (2016).
Riback, J. A. et al. Composition-dependent thermodynamics of intracellular phase separation. Nature 581, 209–214 (2020).
Elbaum-Garfinkle, S. et al. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. Proc. Natl Acad. Sci. USA 112, 7189–7194 (2015).
Hondele, M. et al. DEAD-box ATPases are global regulators of phase-separated organelles. Nature 573, 144–148 (2019).
Nott, T. J. et al. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol. Cell 57, 936–947 (2015). This study is among the first systematic biophysical investigations into the sequence determinants of phase separation as driven by IDRs.
Sankaranarayanan, M. et al. Adaptable P body physical states differentially regulate bicoid mRNA storage during early Drosophila development. Dev. Cell 56, 2886–2901.e6 (2021).
Whitman, B. T., Wang, Y., Murray, C. R. A., Glover, M. J. N. & Owttrim, G. W. Liquid–liquid phase separation of the DEAD-box cyanobacterial RNA helicase redox (CrhR) into dynamic membraneless organelles in Synechocystis sp. strain PCC 6803. Appl. Environ. Microbiol. 89, e0001523 (2023).
Li, Q. et al. DEAD-box helicases modulate dicing body formation in Arabidopsis. Sci. Adv. 7, eabc6266 (2021).
Iserman, C. et al. Condensation of Ded1p promotes a translational switch from housekeeping to stress protein production. Cell 181, 818–831.e19 (2020).
Brady, J. P. et al. Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation. Proc. Natl Acad. Sci. USA 114, E8194–E8203 (2017).
Joseph, J. A. et al. Physics-driven coarse-grained model for biomolecular phase separation with near-quantitative accuracy. Nat. Comput. Sci. 1, 732–743 (2021).
Pak, C. W. et al. Sequence determinants of intracellular phase separation by complex coacervation of a disordered protein. Mol. Cell 63, 72–85 (2016).
Han, T. W. et al. Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies. Cell 149, 768–779 (2012).
Kato, M. et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753–767 (2012).
Boeynaems, S. et al. Phase separation of C9orf72 dipeptide repeats perturbs stress granule dynamics. Mol. Cell 65, 1044–1055.e5 (2017).
Sang, D. et al. Condensed-phase signaling can expand kinase specificity and respond to macromolecular crowding. Mol. Cell 82, 3693–3711.e10 (2022).
Sridharan, S. et al. Systematic discovery of biomolecular condensate-specific protein phosphorylation. Nat. Chem. Biol. 18, 1104–1114 (2022).
Milovanovic, D., Wu, Y., Bian, X. & De Camilli, P. A liquid phase of synapsin and lipid vesicles. Science 361, 604–607 (2018).
Kim, T. H. et al. Phospho-dependent phase separation of FMRP and CAPRIN1 recapitulates regulation of translation and deadenylation. Science 365, 825–829 (2019).
Guo, Y. E. et al. Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature 572, 543–548 (2019).
Nott, T. J., Craggs, T. D. & Baldwin, A. J. Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters. Nat. Chem. 8, 569–575 (2016).
Peeples, W. & Rosen, M. K. Mechanistic dissection of increased enzymatic rate in a phase-separated compartment. Nat. Chem. Biol. 17, 693–702 (2021).
Klosin, A. et al. Phase separation provides a mechanism to reduce noise in cells. Science 367, 464–468 (2020).
To, P. et al. Intrinsically disordered regions promote protein refoldability and facilitate retrieval from biomolecular condensates. Preprint at bioRxiv https://doi.org/10.1101/2023.06.25.546465 (2023).
Lasker, K. et al. The material properties of a bacterial-derived biomolecular condensate tune biological function in natural and synthetic systems. Nat. Commun. 13, 5643 (2022).
Dzuricky, M., Rogers, B. A., Shahid, A., Cremer, P. S. & Chilkoti, A. De novo engineering of intracellular condensates using artificial disordered proteins. Nat. Chem. 12, 814–825 (2020).
Chen, D. et al. Integration of light and temperature sensing by liquid–liquid phase separation of phytochrome B. Mol. Cell 82, 3015–3029.e6 (2022).
Boyd-Shiwarski, C. R. et al. WNK kinases sense molecular crowding and rescue cell volume via phase separation. Cell 185, 4488–4506.e20 (2022).
Wang, B. et al. Condensation of SEUSS promotes hyperosmotic stress tolerance in Arabidopsis. Nat. Chem. Biol. 18, 1361–1369 (2022).
Jalihal, A. P. et al. Multivalent proteins rapidly and reversibly phase-separate upon osmotic cell volume change. Mol. Cell 79, 978–990.e5 (2020).
Dorone, Y. et al. A prion-like protein regulator of seed germination undergoes hydration-dependent phase separation. Cell 184, 4284–4298.e27 (2021).
Yoo, H., Triandafillou, C. & Drummond, D. A. Cellular sensing by phase separation: using the process, not just the products. J. Biol. Chem. 294, 7151–7159 (2019).
Boeynaems, S. et al. Spontaneous driving forces give rise to protein–RNA condensates with coexisting phases and complex material properties. Proc. Natl Acad. Sci. USA 116, 7889–7898 (2019).
Fisher, R. S. & Elbaum-Garfinkle, S. Tunable multiphase dynamics of arginine and lysine liquid condensates. Nat. Commun. 11, 4628 (2020).
Woodruff, J. B., Hyman, A. A. & Boke, E. Organization and function of non-dynamic biomolecular condensates. Trends Biochem. Sci. 43, 81–94 (2018).
Bowman, G. R. et al. A polymeric protein anchors the chromosomal origin/ParB complex at a bacterial cell pole. Cell 134, 945–955 (2008).
Ellisman, M. H., McAdams, H. H. & Shapiro, L. Caulobacter PopZ forms a polar subdomain dictating sequential changes in pole composition and function. Mol. Microbiol. 76, 173–189 (2010).
Lasker, K. et al. Selective sequestration of signalling proteins in a membraneless organelle reinforces the spatial regulation of asymmetry in Caulobacter crescentus. Nat. Microbiol. 5, 418–429 (2020).
Sanders, D. W. et al. Competing protein–RNA interaction networks control multiphase intracellular organization. Cell 181, 306–324.e28 (2020).
Bergeron-Sandoval, L.-P. et al. Endocytic proteins with prion-like domains form viscoelastic condensates that enable membrane remodeling. Proc. Natl Acad. Sci. USA 118, e2113789118 (2021).
Mistry, J. et al. Pfam: the protein families database in 2021. Nucleic Acids Res. 49, D412–D419 (2021).
Plitzko, J. M., Schuler, B. & Selenko, P. Structural biology outside the box–inside the cell. Curr. Opin. Struct. Biol. 46, 110–121 (2017).
Theillet, F.-X. et al. Physicochemical properties of cells and their effects on intrinsically disordered proteins (IDPs). Chem. Rev. 114, 6661–6714 (2014).
Theillet, F.-X. et al. Structural disorder of monomeric α-synuclein persists in mammalian cells. Nature 530, 45–50 (2016).
König, I. et al. Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells. Nat. Methods 12, 773–779 (2015).
Buljan, M. et al. Alternative splicing of intrinsically disordered regions and rewiring of protein interactions. Curr. Opin. Struct. Biol. 23, 443–450 (2013).
Buljan, M. et al. Tissue-specific splicing of disordered segments that embed binding motifs rewires protein interaction networks. Mol. Cell 46, 871–883 (2012).
Niklas, K. J., Bondos, S. E., Dunker, A. K. & Newman, S. A. Rethinking gene regulatory networks in light of alternative splicing, intrinsically disordered protein domains, and post-translational modifications. Front. Cell Dev. Biol. 3, 8 (2015).
Zhou, J., Zhao, S. & Dunker, A. K. Intrinsically disordered proteins link alternative splicing and post-translational modifications to complex cell signaling and regulation. J. Mol. Biol. 430, 2342–2359 (2018).
Subramanian, S. & Kumar, S. Evolutionary anatomies of positions and types of disease-associated and neutral amino acid mutations in the human genome. BMC Genomics 7, 306 (2006).
Vacic, V. & Iakoucheva, L. M. Disease mutations in disordered regions — exception to the rule? Mol. Biosyst. 8, 27–32 (2012).
Vacic, V. et al. Disease-associated mutations disrupt functionally important regions of intrinsic protein disorder. PLoS Comput. Biol. 8, e1002709 (2012).
Tsang, B., Pritišanac, I., Scherer, S. W., Moses, A. M. & Forman-Kay, J. D. Phase separation as a missing mechanism for interpretation of disease mutations. Cell 183, 1742–1756 (2020).
Mészáros, B., Hajdu-Soltész, B., Zeke, A. & Dosztányi, Z. Mutations of intrinsically disordered protein regions can drive cancer but lack therapeutic strategies. Biomolecules 11, 381 (2021).
Kim, H. J. et al. Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. Nature 495, 467–473 (2013).
Patel, A. et al. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162, 1066–1077 (2015).
Dao, T. P. et al. Ubiquitin modulates liquid–liquid phase separation of UBQLN2 via disruption of multivalent interactions. Mol. Cell 69, 965–978.e6 (2018).
Banani, S. F. et al. Genetic variation associated with condensate dysregulation in disease. Dev. Cell 57, 1776–1788.e8 (2022).
Mensah, M. A. et al. Aberrant phase separation and nucleolar dysfunction in rare genetic diseases. Nature 614, 564–571 (2023).
Basu, S. et al. Unblending of transcriptional condensates in human repeat expansion disease. Cell 181, 1062-1079 (2020).
Gemayel, R. et al. Variable glutamine-rich repeats modulate transcription factor activity. Mol. Cell 59, 615–627 (2015).
Boeynaems, S. et al. Aberrant phase separation is a common killing strategy of positively charged peptides in biology and human disease. Preprint at bioRxiv https://doi.org/10.1101/2023.03.09.531820 (2023).
Chandra, B. et al. Phase separation mediates NUP98 fusion oncoprotein leukemic transformation. Cancer Discov. 12, 1152–1169 (2022).
Shirnekhi, H. K., Chandra, B. & Kriwacki, R. W. The role of phase-separated condensates in fusion oncoprotein-driven cancers. Annu. Rev. Cancer Biol. 7, 73–91 (2023).
Simonetti, L., Nilsson, J., McInerney, G., Ivarsson, Y. & Davey, N. E. SLiM-binding pockets: an attractive target for broad-spectrum antivirals. Trends Biochem. Sci. 48, 420–427 (2023).
Madhu, P., Davey, N. E. & Ivarsson, Y. How viral proteins bind short linear motifs and intrinsically disordered domains. Essays Biochem. https://doi.org/10.1042/EBC20220047 (2022).
Vitalis, A. & Pappu, R. V. ABSINTH: a new continuum solvation model for simulations of polypeptides in aqueous solutions. J. Comput. Chem. 30, 673–699 (2009).
Piana, S., Robustelli, P., Tan, D., Chen, S. & Shaw, D. E. Development of a force field for the simulation of single-chain proteins and protein–protein complexes. J. Chem. Theory Comput. 16, 2494–2507 (2020).
Robustelli, P., Piana, S. & Shaw, D. E. Developing a molecular dynamics force field for both folded and disordered protein states. Proc. Natl Acad. Sci. USA 115, E4758–E4766 (2018).
Huang, J. et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat. Methods 14, 71–73 (2017).
Best, R. B., Zheng, W. & Mittal, J. Balanced protein–water interactions improve properties of disordered proteins and non-specific protein association. J. Chem. Theory Comput. 10, 5113–5124 (2014).
Romero, P., Obradovic, Z. & Dunker, A. K. Folding minimal sequences: the lower bound for sequence complexity of globular proteins. FEBS Lett. 462, 363–367 (1999).
Romero, O. & Dunker, K. Sequence data analysis for long disordered regions prediction in the Calcineurin family. Genome Inf. Ser. Workshop Genome Inf. 8, 110–124 (1997).
Romero, P., Obradovic, Z., Kissinger, C., Villafranca, J. E. & Dunker, A. K. Identifying disordered regions in proteins from amino acid sequence. In Proc. International Conference on Neural Networks (ICNN’97), Vol. 1, 90–95 (1997).
Uversky, V. N., Gillespie, J. R. & Fink, A. L. Why are ‘natively unfolded’ proteins unstructured under physiologic conditions? Proteins Struct. Funct. Bioinf. 41, 415–427 (2000).
Emenecker, R. J., Griffith, D. & Holehouse, A. S. Metapredict: a fast, accurate, and easy-to-use predictor of consensus disorder and structure. Biophys. J. 120, 4312–4319 (2021).
Gibbs, E. B., Cook, E. C. & Showalter, S. A. Application of NMR to studies of intrinsically disordered proteins. Arch. Biochem. Biophys. 628, 57–70 (2017).
Camacho-Zarco, A. R. et al. NMR provides unique insight into the functional dynamics and interactions of intrinsically disordered proteins. Chem. Rev. 122, 9331–9356 (2022).
Schuler, B., Soranno, A., Hofmann, H. & Nettels, D. Single-molecule FRET spectroscopy and the polymer physics of unfolded and intrinsically disordered proteins. Annu. Rev. Biophys. 45, 207–231 (2016).
Brucale, M., Schuler, B. & Samorì, B. Single-molecule studies of intrinsically disordered proteins. Chem. Rev. 114, 3281–3317 (2014).
Balasubramaniam, D. & Komives, E. A. Hydrogen-exchange mass spectrometry for the study of intrinsic disorder in proteins. Biochim. Biophys. Acta 1834, 1202–1209 (2013).
Leeb, S. & Danielsson, J. Obtaining hydrodynamic radii of intrinsically disordered protein ensembles by pulsed field gradient NMR measurements. Methods Mol. Biol. 2141, 285–302 (2020).
Fuertes, G. et al. Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements. Proc. Natl Acad. Sci. USA 114, E6342–E6351 (2017).
Kikhney, A. G. & Svergun, D. I. A practical guide to small angle X‐ray scattering (SAXS) of flexible and intrinsically disordered proteins. FEBS Lett. 589, 2570–2577 (2015).
Martin, E. W., Hopkins, J. B. & Mittag, T. Small-angle X-ray scattering experiments of monodisperse intrinsically disordered protein samples close to the solubility limit. Methods Enzymol. 646, 185–222 (2021).
Riback, J. A. et al. Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water. Science 358, 238–241 (2017).
Cubuk, J., Stuchell-Brereton, M. D. & Soranno, A. The biophysics of disordered proteins from the point of view of single-molecule fluorescence spectroscopy. Essays Biochem. 66, 875–890 (2022).
Chemes, L. B., Alonso, L. G., Noval, M. G. & de Prat-Gay, G. Circular dichroism techniques for the analysis of intrinsically disordered proteins and domains. Methods Mol. Biol. 895, 387–404 (2012).
Stuchfield, D. & Barran, P. Unique insights to intrinsically disordered proteins provided by ion mobility mass spectrometry. Curr. Opin. Chem. Biol. 42, 177–185 (2018).
Kassem, N. et al. Order and disorder — an integrative structure of the full-length human growth hormone receptor. Sci. Adv. 7, eabh3805 (2021).
Borgia, A. et al. Consistent view of polypeptide chain expansion in chemical denaturants from multiple experimental methods. J. Am. Chem. Soc. 138, 11714–11726 (2016).
Zheng, W. et al. Probing the action of chemical denaturant on an intrinsically disordered protein by simulation and experiment. J. Am. Chem. Soc. 138, 11702–11713 (2016).
Naudi-Fabra, S., Tengo, M., Jensen, M. R., Blackledge, M. & Milles, S. Quantitative description of intrinsically disordered proteins using single-molecule FRET, NMR, and SAXS. J. Am. Chem. Soc. 143, 20109–20121 (2021).
Clark, P. L., Plaxco, K. W. & Sosnick, T. R. Water as a good solvent for unfolded proteins: folding and collapse are fundamentally different. J. Mol. Biol. 432, 2882–2889 (2020).
Best, R. B. Emerging consensus on the collapse of unfolded and intrinsically disordered proteins in water. Curr. Opin. Struct. Biol. 60, 27–38 (2020).
Guseva, S. et al. Measles virus nucleo- and phosphoproteins form liquid-like phase-separated compartments that promote nucleocapsid assembly. Sci. Adv. 6, eaaz7095 (2020). Along with Milles et al. (2018), this paper provides the biophysical basis for weak multivalent interactions that underlie how viral proteins can mediate condensate formation.
Shea, J.-E., Best, R. B. & Mittal, J. Physics-based computational and theoretical approaches to intrinsically disordered proteins. Curr. Opin. Struct. Biol. 67, 219–225 (2021).
Zerze, G. H., Best, R. B. & Mittal, J. Sequence- and temperature-dependent properties of unfolded and disordered proteins from atomistic simulations. J. Phys. Chem. B 119, 14622–14630 (2015).
Zerze, G. H., Zheng, W., Best, R. B. & Mittal, J. Evolution of all-atom protein force fields to improve local and global properties. J. Phys. Chem. Lett. 10, 2227–2234 (2019).
Piana, S., Donchev, A. G., Robustelli, P. & Shaw, D. E. Water dispersion interactions strongly influence simulated structural properties of disordered protein states. J. Phys. Chem. B 119, 5113–5123 (2015).
Huang, J. & MacKerell, A. D. Jr. Force field development and simulations of intrinsically disordered proteins. Curr. Opin. Struct. Biol. 48, 40–48 (2018).
Tesei, G., Schulze, T. K., Crehuet, R. & Lindorff-Larsen, K. Accurate model of liquid–liquid phase behavior of intrinsically disordered proteins from optimization of single-chain properties. Proc. Natl Acad. Sci. USA 118, e2111696118 (2021).
Palazzesi, F., Prakash, M. K., Bonomi, M. & Barducci, A. Accuracy of current all-atom force-fields in modeling protein disordered states. J. Chem. Theory Comput. 11, 2–7 (2015).
Baul, U., Chakraborty, D., Mugnai, M. L., Straub, J. E. & Thirumalai, D. Sequence effects on size, shape, and structural heterogeneity in intrinsically disordered proteins. J. Phys. Chem. B 123, 3462–3474 (2019).
Dignon, G. L., Zheng, W., Kim, Y. C., Best, R. B. & Mittal, J. Sequence determinants of protein phase behavior from a coarse-grained model. PLoS Comput. Biol. 14, e1005941 (2018).
Ozenne, V. et al. Flexible-meccano: a tool for the generation of explicit ensemble descriptions of intrinsically disordered proteins and their associated experimental observables. Bioinformatics 28, 1463–1470 (2012).
Tria, G., Mertens, H. D. T., Kachala, M. & Svergun, D. I. Advanced ensemble modelling of flexible macromolecules using X-ray solution scattering. IUCrJ 2, 207–217 (2015).
Nodet, G. et al. Quantitative description of backbone conformational sampling of unfolded proteins at amino acid resolution from NMR residual dipolar couplings. J. Am. Chem. Soc. 131, 17908–17918 (2009).
Bottaro, S., Bengtsen, T. & Lindorff-Larsen, K. Integrating molecular simulation and experimental data: a Bayesian/maximum entropy reweighting approach. Methods Mol. Biol. 2112, 219–240 (2018).
Brookes, D. H. & Head-Gordon, T. Experimental inferential structure determination of ensembles for intrinsically disordered proteins. J. Am. Chem. Soc. 138, 4530–4538 (2016).
Leung, H. T. A. et al. A rigorous and efficient method to reweight very large conformational ensembles using average experimental data and to determine their relative information content. J. Chem. Theory Comput. 12, 383–394 (2016).
Bonomi, M., Camilloni, C., Cavalli, A. & Vendruscolo, M. Metainference: a Bayesian inference method for heterogeneous systems. Sci. Adv. 2, e1501177 (2016).
Zhang, O. et al. Learning to evolve structural ensembles of unfolded and disordered proteins using experimental solution data. J. Chem. Phys. 158, 174113 (2023).
Bonomi, M., Heller, G. T., Camilloni, C. & Vendruscolo, M. Principles of protein structural ensemble determination. Curr. Opin. Struct. Biol. 42, 106–116 (2017).
Thomasen, F. E. & Lindorff-Larsen, K. Conformational ensembles of intrinsically disordered proteins and flexible multidomain proteins. Biochem. Soc. Trans. 50, 541–554 (2022).
Chen, J. W., Romero, P., Uversky, V. N. & Dunker, A. K. Conservation of intrinsic disorder in protein domains and families: I. A database of conserved predicted disordered regions. J. Proteome Res. 5, 879–887 (2006).
Chen, J. W., Romero, P., Uversky, V. N. & Dunker, A. K. Conservation of intrinsic disorder in protein domains and families. II. Functions of conserved disorder. J. Proteome Res. 5, 888–898 (2006).
Shinn, M. K. et al. Connecting sequence features within the disordered C-terminal linker of Bacillus subtilis FtsZ to functions and bacterial cell division. Proc. Natl Acad. Sci. USA 119, e2211178119 (2022).
Pancsa, R., Zsolyomi, F. & Tompa, P. Co-evolution of intrinsically disordered proteins with folded partners witnessed by evolutionary couplings. Int. J. Mol. Sci. 19, 3315 (2018).
Toth-Petroczy, A. et al. Structured states of disordered proteins from genomic sequences. Cell 167, 158–170.e12 (2016).
Hsu, I. S. et al. A functionally divergent intrinsically disordered region underlying the conservation of stochastic signaling. PLoS Genet. 17, e1009629 (2021).
Hwang, T. et al. Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP protein ENAH. eLife 11, e70680 (2022).
Hwang, T. et al. A distributed residue network permits conformational binding specificity in a conserved family of actin remodelers. eLife 10, e70601 (2021).
Markin, C. J. et al. Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics. Science 373, eabf8761 (2021).
Pincus, D. et al. Engineering allosteric regulation in protein kinases. Sci. Signal. 11, eaar3250 (2018).
Acknowledgements
The authors thank R. Pappu for discussions in the initial phase of writing; G. Daughdrill, A. Flynn, J. Forman-Kay, P. Jemth, A. Moses, J.G. Olsen, R. Pappu, B. Schuler, K. Skriver and S. Sukenik for valuable comments and suggestions; and S. Boeynaems for original microscopy images in Fig. 5. This work was supported by the Novo Nordisk Foundation challenge grant REPIN, rethinking protein interactions (NNF18OC0033926 to B.B.K.), by the Danish Research Councils (9040-00164B to B.B.K.), by the United States National Science Foundation (NSF) (NSF 2128068 to A.S.H.), by the US NIH (DP2 CA290639-01 to A.S.H.) and by the Human Frontiers in Science Program (RGP0015/2022 to A.S.H.).
Author information
Authors and Affiliations
Contributions
The authors contributed equally to all aspects of the article.
Corresponding authors
Ethics declarations
Competing interest
A.S.H. is a scientific consultant with Dewpoint Therapeutics and on the Scientific Advisory Board for Prose Foods. B.B.K. declares no conflicts of interest.
Peer review
Peer review information
Nature Reviews Molecular Cell Biology thanks Elizabeth Rhoades and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Related links
CAID prediction portal: https://caid.idpcentral.org/submit
CIDER webserver for calculating sequence properties: http://pappulab.wustl.edu/CIDER/
Eukaryotic Linear Motif (ELM) resource: http://elm.eu.org/
Link to bioinformatic analysis referred to in this paper: https://github.com/holehouse-lab/supportingdata/tree/master/2023/holehouse_and_kragelund_2023
Metapredict disorder predictor: https://metapredict.net/
PLAAC webserver for identify prion-like domains: http://plaac.wi.mit.edu/
Supplementary information
41580_2023_673_MOESM1_ESM.mp4
Movie S1 Rendering of all-atom simulation of the hnRNPA1 IDR to illustrate the conformational heterogeneity within an atomistic ensemble57. Conformations were generated through all-atom Monte Carlo simulations, which show good agreement with experimental characterization.
Glossary
- π–π interactions
-
Interactions mediated by delocalized π electron clouds, seen in amino acids with aromatic side chains.
- Associative polymers
-
A class of polymer architecture in which specific regions or monomers contribute associated (attractive) interactions. See foundational work by Cate and Whitten (1986) and Semenov and Rubinstein (1998).
- Conformational selection
-
A mode of binding in which the intrinsically disordered region binds to a partner by adopting a binding-competent conformation in the unbound ensemble, which then binds without further conformational re-arrangement. Unlike induced fit, the bound-state configuration of the intrinsically disordered region is visited in the unbound ensemble, such that the binding partner ‘selects’ a specific conformation to bind.
- Deep learning
-
Deep learning is a branch of machine learning concerned with models that contain large numbers of parameters. It has received substantial attention owing to its ability to perform complex pattern recognition, especially for text and images. In the biological sciences, deep learning has been applied to protein structure prediction, disorder prediction and, more recently, the prediction of ensemble properties.
- End-to-end distance
-
Also written as Re. This parameter is a measure of global ensemble dimensions and reports on the average distance between the first and last residues in the intrinsically disordered region.
- Forcefields
-
In molecular simulations, forcefields are the set of equations and parameters used to describe the chemical physics of the molecular system of interest. All-atom forcefields used for simulating disordered proteins include ABSINTH, amber03ws, a99SB-disp, CHARMM36m and DES-Amber391,392,393,394,395.
- Hydrodynamic radius
-
Also written as Rh. This parameter is a measure of global ensemble dimensions and reports on the radius associated with a sphere that would diffuse through the solution at the same speed the intrinsically disordered region in question would, after correcting for solution viscosity.
- Induced fit
-
A mode of binding in which the intrinsically disordered region is templated into a specific conformation by a binding partner. Unlike conformational selection, the bound-state conformation of the intrinsically disordered region is never/rarely visited in the unbound ensemble, and the act of binding ‘induces’ this bound-state conformation.
- Molecular grammar
-
When used in the context of intrinsically disordered regions and biomolecular condensates, this refers to the grammar of sequence features that dictate the driving forces for condensate formation and the resulting material properties.
- Prion-like domains
-
(PLDs). A class of protein domains defined by being of low complexity (many similar amino acids) and possessing enrichment for polar amino acids (especially glutamine, asparagine, glycine and serine), often with additional aromatic residues. PLDs are defined using the PLAAC webserver with default parameters. Although PLDs have been found to phase separate, their presence should not be taken as evidence that a protein will phase separate. They are named after yeast prions, in which a PLD was originally defined.
- Radius of gyration
-
Also written as Rg. This parameter is a measure of global ensemble dimensions and reports on the average distance between the centre of mass of the intrinsically disordered region and the individual atoms.
- Sequence features
-
Properties of an intrinsically disordered region amino acid sequence that are determined by the composition and patterning of different amino acids. Sequence features can — by definition — be determined directly from sequence. Several commonly used sequence features can be calculated using the CIDER webserver.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Holehouse, A.S., Kragelund, B.B. The molecular basis for cellular function of intrinsically disordered protein regions. Nat Rev Mol Cell Biol 25, 187–211 (2024). https://doi.org/10.1038/s41580-023-00673-0
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41580-023-00673-0
This article is cited by
-
DNA binding redistributes activation domain ensemble and accessibility in pioneer factor Sox2
Nature Communications (2024)
-
Conformational ensembles of the human intrinsically disordered proteome
Nature (2024)
-
The acidic intrinsically disordered region of the inflammatory mediator HMGB1 mediates fuzzy interactions with CXCL12
Nature Communications (2024)
-
Direct prediction of intrinsically disordered protein conformational properties from sequence
Nature Methods (2024)
-
An easy-to-use computational tool for predicting 3D properties of disordered proteins
Nature Methods (2024)
