bims-indpro Biomed News
on Intrinsically disordered proteins
Issue of 2022–05–22
fourteen papers selected by
Sara Mingu, Johannes Gutenberg University



  1. ACS Macro Lett. 2022 Mar 15. 11(3): 382-386
      Overall charged polymers with quenched charge sequences often adopt partially globular structures which result from the interplay between the disorder in charge sequences and thermal fluctuations. Simple energetic considerations show that structures consisting of alike (equal-size-equal-charge) globules are not favorable: the structures are intrinsically heterogeneous. We predict the globule distributions with the lowest energies in the size-charge space. The favorable structures comprise large (undercharged) and a majority of small (overcharged) globules. These distributions build a well characterized compact subset, which suggests some order. We also perform large scale molecular dynamics simulations on random quenched +/- sequences. Simulation results show that, despite disorder, the random charge sequences preferentially visit the predicted low energy structures and the predicted order emerges in the pearl-size distribution. This good agreement validates a posteriori the simple expression used for the energy. Implications for polyampholytes, polyelectrolytes, and intrinsically disordered proteins are discussed.
    DOI:  https://doi.org/10.1021/acsmacrolett.1c00655
  2. ACS Macro Lett. 2022 Apr 19. 11(4): 562-567
      Liquid-liquid phase separation (LLPS) emerges as a fundamental underlying mechanism for the biological organization, especially the formation of membraneless organelles (MLOs) hosting intrinsically disordered proteins (IDPs) as scaffolds. Nucleic acids are compositional biomacromolecules of MLOs with wide implications in normal cell functions as well as in pathophysiology caused by aberrant phase behavior. Exploiting a minimalist artificial membraneless organelles (AMLO) from LLPS of IDP-mimicking polymer-oligopeptide hybrid (IPH), we investigated the effect of nucleic acids with different lengths and sequence variations on AMLO. The behavior of this AMLO in the presence of DNAs and RNAs resembled natural MLOs in multiple aspects, namely, modulated propensity of formation, morphology, liquidity, and dynamics. Both DNA and RNA could enhance the LLPS of AMLO, while compared with RNA, DNA had a higher tendency to solidify and diminish dynamics thereof. These findings suggest its potential as a concise model system for the understanding of the interaction between nucleic acids and natural MLOs and for studying the molecular mechanism of diseases involving MLOs.
    DOI:  https://doi.org/10.1021/acsmacrolett.2c00167
  3. Front Mol Biosci. 2022 ;9 862910
      Human epidermal growth factor receptors (HER/ERBB) form dimers that promote cell proliferation, migration, and differentiation, but overexpression of HER proteins results in cancer. Consequently, inhibitors of HER dimerization may function as effective antitumor drugs. An alternatively spliced variant of HER2, called herstatin, is an autoinhibitor of HER proteins, and the intron 8-encoded 79-residue domain of herstatin, called Int8, binds HER family receptors even in isolation. However, the structure of Int8 remains poorly understood. Here, we revealed by circular dichroism, NMR, small-angle X-ray scattering, and structure prediction that isolated Int8 is largely disordered but has a residual helical structure. The radius of gyration of Int8 was almost the same as that of fully unfolded states, although the conformational ensemble of Int8 was less flexible than random coils. These results demonstrate that Int8 is intrinsically disordered. Thus, Int8 is an interesting example of an intrinsically disordered region with tumor-suppressive activity encoded by an intron. Furthermore, we show that the R371I mutant of Int8, which is defective in binding to HER2, is prone to aggregation, providing a rationale for the loss of function.
    Keywords:  herstatin; human epidermal growth factor receptor; intrinsically disordered protein; intron-encoded protein; pre-molten globule state; small-angle X-ray scattering
    DOI:  https://doi.org/10.3389/fmolb.2022.862910
  4. Adv Sci (Weinh). 2022 May 18. e2201444
      The slime of velvet worms (Onychophora) is a strong and fully biodegradable protein material, which upon ejection undergoes a fast liquid-to-solid transition to ensnare prey. However, the molecular mechanisms of slime self-assembly are still not well understood, notably because the primary structures of slime proteins are yet unknown. Combining transcriptomic and proteomic studies, the complete primary sequences of slime proteins are obtained and identified key features for slime self-assembly. The high molecular weight slime proteins contain cysteine residues at the N- and C-termini that mediate the formation of multi-protein complexes via disulfide bonding. Low complexity domains in the N-termini are also identified and their propensity for liquid-liquid phase separation is established, which may play a central role in slime biofabrication. Using solid-state nuclear magnetic resonance, rigid and flexible domains of the slime proteins are mapped to specific peptide domains. The complete sequencing of major slime proteins is an important step toward sustainable fabrication of polymers inspired by the velvet worm slime.
    Keywords:  fibers; nuclear magnetic resonance; protein sequence; proteomics; slime; structure; velvet worms
    DOI:  https://doi.org/10.1002/advs.202201444
  5. Front Mol Biosci. 2022 ;9 863141
      Intrinsically disordered proteins lack a stable tertiary structure and form dynamic conformational ensembles due to their characteristic physicochemical properties and amino acid composition. They are abundant in nature and responsible for a large variety of cellular functions. While numerous bioinformatics tools have been developed for in silico disorder prediction in the last decades, there is a need for experimental methods to verify the disordered state. CD spectroscopy is widely used for protein secondary structure analysis. It is usable in a wide concentration range under various buffer conditions. Even without providing high-resolution information, it is especially useful when NMR, X-ray, or other techniques are problematic or one simply needs a fast technique to verify the structure of proteins. Here, we propose an automatized binary disorder-order classification method by analyzing far-UV CD spectroscopy data. The method needs CD data at only three wavelength points, making high-throughput data collection possible. The mathematical analysis applies the k-nearest neighbor algorithm with cosine distance function, which is independent of the spectral amplitude and thus free of concentration determination errors. Moreover, the method can be used even for strong absorbing samples, such as the case of crowded environmental conditions, if the spectrum can be recorded down to the wavelength of 212 nm. We believe the classification method will be useful in identifying disorder and will also facilitate the growth of experimental data in IDP databases. The method is implemented on a webserver and freely available for academic users.
    Keywords:  CD spectroscopy; disorder identifier; disorder–order classification; intrinsically disordered proteins; machine learning; protein secondary structure
    DOI:  https://doi.org/10.3389/fmolb.2022.863141
  6. Curr Protein Pept Sci. 2022 May 18.
       BACKGROUND: Clostridiodes (or Clostridium) difficile is a spore-forming, Gram-positive anaerobic bacterium which may cause symptoms ranging from diarrhea to pseudomembranous colitis. During the C. difficile infection (CDI), the two primary bacterial toxins, toxin A (TcdA) or toxin B (TcdB), disrupt host cell function mainly through the inactivation of small GTPases that regulate the actin cytoskeleton. Both toxins have complex structural organization containing several functional domains.
    METHODS: Analytical bioinformatics tools are used to compare the extent of disorder within TcdA and TcdB proteins, and to see if the existence of structural disorder can be used to explain the difference in the functionality of these toxins.
    RESULTS: This paper's aim is to offer an overall review of the structural and functional differences between TcdA and TcdB.
    CONCLUSIONS: Results of our multifactorial bioinformatics analysis revealed that intrinsic disorder may play a role in the multifunctionality of C. difficile major toxins TcdA and TcdB, suggesting that intrinsic disorder may be related to their pathogenic mechanisms.
    Keywords:  TcdA; TcdB; bacterial toxin; binding promiscuity; intrinsic disorder; multifunctionality; protein-protein interactions
    DOI:  https://doi.org/10.2174/1389203723666220518111801
  7. Nat Commun. 2022 May 20. 13(1): 2818
      Molecular chaperones play a central role in regulating protein homeostasis, and their active forms often contain intrinsically disordered regions (IDRs). However, how IDRs impact chaperone action remains poorly understood. Here, we discover that the disordered N terminus of the prototype chaperone Spy facilitates client release. With NMR spectroscopy and molecular dynamics simulations, we find that the N terminus can bind transiently to the client-binding cavity of Spy primarily through electrostatic interactions mediated by the N-terminal D26 residue. This intramolecular interaction results in a dynamic competition of the N terminus with the client for binding to Spy, which promotes client discharge. Our results reveal the mechanism by which Spy releases clients independent of energy input, thus enriching the current knowledge on how ATP-independent chaperones release their clients and highlighting the importance of synergy between IDRs and structural domains in regulating protein function.
    DOI:  https://doi.org/10.1038/s41467-022-30499-x
  8. Genomics. 2022 May 16. pii: S0888-7543(22)00135-5. [Epub ahead of print] 110390
      NF-Y is a trimeric pioneer Transcription Factor (TF) whose target sequence -the CCAAT box- is present in ~25% of mammalian promoters. We reconstruct the phylogenetic history of the regulatory NF-YA subunit in vertebrates. We find that in addition to the remarkable conservation of the subunits-interaction and DNA-binding parts, the Transcriptional Activation Domain (TAD) is also conserved (>90% identity among bony vertebrates). We infer the phylogeny of the alternatively spliced exon-3 and partial splicing events of exon-7 -7N and 7C- revealing independent clade-specific losses of these regions. These isoforms shape the TAD. Absence of exon-3 in basal deuterostomes, cartilaginous fishes and hagfish, but not in lampreys, suggests that the "short" isoform is primordial, with emergence of exon-3 in chordates. Exon 7N was present in the vertebrate common ancestor, while 7C is a molecular innovation of teleost fishes. RNA-seq analysis in several species confirms expression of all these isoforms. We identify 3 blocks of amino acids in the TAD shared across deuterostomes, yet structural predictions and sequence analyses suggest an evolutionary drive for maintenance of an Intrinsically Disordered Region -IDR- within the TAD. Overall, these data help reconstruct the logic for alternative splicing of this essential eukaryotic TF.
    Keywords:  Alternative splicing; Evolution; Glutamine-rich; Intrinsically disordered region; NFYA; Transactivation domain; Transcription factor
    DOI:  https://doi.org/10.1016/j.ygeno.2022.110390
  9. Mol Cell. 2022 May 10. pii: S1097-2765(22)00385-9. [Epub ahead of print]
      Sex differences are pervasive in human health and disease. One major key to sex-biased differences lies in the sex chromosomes. Although the functions of the X chromosome proteins are well appreciated, how they compare with their Y chromosome homologs remains elusive. Herein, using ensemble and single-molecule techniques, we report that the sex chromosome-encoded RNA helicases DDX3X and DDX3Y are distinct in their propensities for liquid-liquid phase separation (LLPS), dissolution, and translation repression. We demonstrate that the N-terminal intrinsically disordered region of DDX3Y more strongly promotes LLPS than the corresponding region of DDX3X and that the weaker ATPase activity of DDX3Y, compared with DDX3X, contributes to the slower disassembly dynamics of DDX3Y-positive condensates. Interestingly, DDX3Y-dependent LLPS represses mRNA translation and enhances aggregation of FUS more strongly than DDX3X-dependent LLPS. Our study provides a platform for future comparisons of sex chromosome-encoded protein homologs, providing insights into sex differences in RNA metabolism and human disease.
    Keywords:  ATPase activity; DDX3X; DDX3Y; RNA helicase; condensates; liquid-liquid phase separation; sex chromosome homolog proteins; translation repression
    DOI:  https://doi.org/10.1016/j.molcel.2022.04.022
  10. PLoS Biol. 2022 May 16. 20(5): e3001636
      The recent revolution in computational protein structure prediction provides folding models for entire proteomes, which can now be integrated with large-scale experimental data. Mass spectrometry (MS)-based proteomics has identified and quantified tens of thousands of posttranslational modifications (PTMs), most of them of uncertain functional relevance. In this study, we determine the structural context of these PTMs and investigate how this information can be leveraged to pinpoint potential regulatory sites. Our analysis uncovers global patterns of PTM occurrence across folded and intrinsically disordered regions. We found that this information can help to distinguish regulatory PTMs from those marking improperly folded proteins. Interestingly, the human proteome contains thousands of proteins that have large folded domains linked by short, disordered regions that are strongly enriched in regulatory phosphosites. These include well-known kinase activation loops that induce protein conformational changes upon phosphorylation. This regulatory mechanism appears to be widespread in kinases but also occurs in other protein families such as solute carriers. It is not limited to phosphorylation but includes ubiquitination and acetylation sites as well. Furthermore, we performed three-dimensional proximity analysis, which revealed examples of spatial coregulation of different PTM types and potential PTM crosstalk. To enable the community to build upon these first analyses, we provide tools for 3D visualization of proteomics data and PTMs as well as python libraries for data accession and processing.
    DOI:  https://doi.org/10.1371/journal.pbio.3001636
  11. J Cell Sci. 2022 May 19. pii: jcs.259893. [Epub ahead of print]
      In Trypanosoma brucei, transition fibres (TF) form a nine-bladed pattern-like structure connecting the base of the flagellum to the flagellar pocket membrane. Despite the characterization of two TF proteins, CEP164C and TbRP2, little is known about the organization of these fibres. Here, we report the identification and characterization of the first kinetoplastid-specific TF protein named TFK1 (Tb927.6.1180). Bioinformatics and functional domain analysis identified three TFK1 distinct domains: an N-terminal domain of an unpredicted function, a coiled-coil domain involved in TFK1-TFK1 interaction and a C-terminal intrinsically disordered region potentially involved in protein interaction. Cellular immuno-localization showed that TFK1 is a newly identified basal body maturation marker. Further, using ultrastructure expansion and immuno-electron microscopies we localized CEP164C and TbRP2 at the TF and TFK1 on the distal appendage matrix of the TF. Importantly, RNAi knockdown of TFK1 in bloodstream form cells induced misplacement of basal bodies, a defect in the furrow or fold generation and eventually cell death. We hypothesize that TFK1 is a basal body positioning specific actor and a key regulator of cytokinesis in the bloodstream form Trypanosoma brucei.
    Keywords:  Basal body; Cytokinesis; Flagellum; Transition Fibres protein Kinetoplastid specific-1; Transition fibres; Trypanosoma brucei
    DOI:  https://doi.org/10.1242/jcs.259893
  12. Nat Commun. 2022 May 18. 13(1): 2724
      Oncoprotein SS18-SSX is a hallmark of synovial sarcomas. However, as a part of the SS18-SSX fusion protein, SS18's function remains unclear. Here, we depict the structures of both human SS18/BRG1 and yeast SNF11/SNF2 subcomplexes. Both subcomplexes assemble into heterodimers that share a similar conformation, suggesting that SNF11 might be a homologue of SS18 in chromatin remodeling complexes. Importantly, our study shows that the self-association of the intrinsically disordered region, QPGY domain, leads to liquid-liquid phase separation (LLPS) of SS18 or SS18-SSX and the subsequent recruitment of BRG1 into phase-separated condensates. Moreover, our results show that the tyrosine residues in the QPGY domain play a decisive role in the LLPS of SS18 or SS18-SSX. Perturbations of either SS18-SSX LLPS or SS18-SSX's binding to BRG1 impair NIH3T3 cell transformation by SS18-SSX. Our data demonstrate that both LLPS and assembling into chromatin remodelers contribute to the oncogenic activity of SS18-SSX in synovial sarcomas.
    DOI:  https://doi.org/10.1038/s41467-022-30447-9
  13. Front Plant Sci. 2022 ;13 886525
      Plants can be exposed to cold temperatures and have therefore evolved several mechanisms to prevent damage caused by freezing. One of the most important targets are membranes, which are particularly susceptible to cold damage. To protect against such abiotic stresses, plants express a family of proteins known as late embryogenesis abundant (LEA) proteins. Many LEA proteins are intrinsically disordered, that is, they do not contain stable secondary or tertiary structures alone in solution. These proteins have been shown in a number of studies to protect plants from damage caused by cold, drought, salinity, and osmotic stress. In this family, the most studied proteins are the type II LEA proteins, better known as dehydrins (dehydration-induced proteins). Many physiological studies have shown that dehydrins are often located near the membrane during abiotic stress and that the expression of dehydrins helps to prevent the formation of oxidation-modified lipids and reduce the amount of electrolyte leakage, two hallmarks of damaged membranes. One of the earliest biophysical clues that dehydrins are involved in membrane cryoprotection came from in vitro studies that demonstrated a binding interaction between the protein and membranes. Subsequent work has shown that one conserved motif, known as K-segments, is involved in binding, while recent studies have used NMR to explore the residue specific structure of dehydrins when bound to membranes. The biophysical techniques also provide insight into the mechanism by which dehydrins protect the membrane from cold stress, which appears to mainly involve the lowering of the transition temperature.
    Keywords:  abiotic stress; dehydrins; intrinsically disordered proteins; membranes; physiological response; structure
    DOI:  https://doi.org/10.3389/fpls.2022.886525
  14. J Biol Chem. 2022 May 14. pii: S0021-9258(22)00472-0. [Epub ahead of print] 102032
      CARD8 is a pattern-recognition receptor that forms a caspase-1-activating inflammasome. CARD8 undergoes constitutive autoproteolysis, generating an N-terminal (NT) fragment with a disordered region and a ZU5 domain and a C-terminal (CT) fragment with UPA and CARD domains. Dipeptidyl peptidase (DPP) 8 and DPP9 inhibitors, including Val-boroPro (VbP), accelerate the degradation of the NT fragment via a poorly characterized proteasome-mediated pathway, thereby releasing the inflammatory CT fragment from autoinhibition. Here, we show that the core 20S proteasome, which degrades disordered and misfolded proteins independent of ubiquitin modification, controls activation of the CARD8 inflammasome. In unstressed cells, we discovered that the 20S proteasome degrades just the NT disordered region, leaving behind the folded ZU5, UPA, and CARD domains to act as an inhibitor of inflammasome assembly. However, in VbP-stressed cells, we show the 20S proteasome degrades the entire NT fragment, perhaps due to ZU5 domain unfolding, freeing the CT fragment from autoinhibition. Taken together, these results show that the susceptibility of the CARD8 NT domain to 20S proteasome-mediated degradation controls inflammasome activation.
    Keywords:  CARD8; DPP8/9; inflammasomes; proteasome; protein disorder; ubiquitin
    DOI:  https://doi.org/10.1016/j.jbc.2022.102032