bims-indpro Biomed News
on Intrinsically disordered proteins
Issue of 2022‒11‒13
sixteen papers selected by
Sara Mingu
Johannes Gutenberg University


  1. Essays Biochem. 2022 Nov 09. pii: EBC20220052. [Epub ahead of print]
      The facts that many proteins with crucial biological functions do not have unique structures and that many biological processes are compartmentalized into the liquid-like biomolecular condensates, which are formed via liquid-liquid phase separation (LLPS) and are not surrounded by the membrane, are revolutionizing the modern biology. These phenomena are interlinked, as the presence of intrinsic disorder represents an important requirement for a protein to undergo LLPS that drives biogenesis of numerous membrane-less organelles (MLOs). Therefore, one can consider these phenomena as crucial constituents of a new IDP-LLPS-MLO field. Furthermore, intrinsically disordered proteins (IDPs), LLPS, and MLOs represent a clear link between molecular and cellular biology and soft matter and condensed soft matter physics. Both IDP and LLPS/MLO fields are undergoing explosive development and generate the ever-increasing mountain of crucial data. These new data provide answers to so many long-standing questions that it is difficult to imagine that in the very recent past, protein scientists and cellular biologists operated without taking these revolutionary concepts into account. The goal of this essay is not to deliver a comprehensive review of the IDP-LLPS-MLO field but to provide a brief and rather subjective outline of some of the recent developments in these exciting fields.
    Keywords:  Intrinsically disordered protein; Liquid-liquid phase separation; Membraneless organelle; Protein-protein interactions; Soft matter; Water structure
    DOI:  https://doi.org/10.1042/EBC20220052
  2. Sci Rep. 2022 Nov 09. 12(1): 19057
      Intrinsically disordered proteins (IDP) are at the center of numerous biological processes, and attract consequently extreme interest in structural biology. Numerous approaches have been developed for generating sets of IDP conformations verifying a given set of experimental measurements. We propose here to perform a systematic enumeration of protein conformations, carried out using the TAiBP approach based on distance geometry. This enumeration was performed on two proteins, Sic1 and pSic1, corresponding to unphosphorylated and phosphorylated states of an IDP. The relative populations of the obtained conformations were then obtained by fitting SAXS curves as well as Ramachandran probability maps, the original finite mixture approach RamaMix being developed for this second task. The similarity between profiles of local gyration radii provides to a certain extent a converged view of the Sic1 and pSic1 conformational space. Profiles and populations are thus proposed for describing IDP conformations. Different variations of the resulting gyration radius between phosphorylated and unphosphorylated states are observed, depending on the set of enumerated conformations as well as on the methods used for obtaining the populations.
    DOI:  https://doi.org/10.1038/s41598-022-21648-9
  3. Essays Biochem. 2022 Nov 09. pii: EBC20220054. [Epub ahead of print]
      Biomolecular condensates are functional assemblies, which can enrich intrinsically disordered proteins (IDPs) and/or RNAs at concentrations that are orders of magnitude higher than the bulk. In their native functional state, these structures can exist in multiple physical states including liquid-droplet phase, hydrogels, and solid assemblies. On the other hand, an aberrant transition between these physical states can result in loss-of-function or a gain-of-toxic-function. A prime example of such an aberrant transition is droplet aging-a phenomenon where some condensates may progressively transition into less dynamic material states at biologically relevant timescales. In this essay, we review structural and viscoelastic roots of aberrant liquid-solid transitions. Also, we highlight the different checkpoints and experimentally tunable handles, both active (ATP-dependent enzymes, post-translational modifications) and passive (colocalization of RNA molecules), that could alter the material state of assemblies.
    Keywords:  LLPS; condensate aging; condensates; liquid-solid transition; phase separation; post-translational modifications
    DOI:  https://doi.org/10.1042/EBC20220054
  4. Nano Lett. 2022 Nov 08.
      Understanding the interactions between nanoparticles (NPs) and proteins is crucial for the successful application of NPs in biological contexts. Protein adsorption is dependent on particle size, and protein binding to ultrasmall (1-3 nm) NPs is considered to be generally weak. However, most studies have involved structured biomacromolecules, while the interactions of ultrasmall NPs with intrinsically disordered proteins (IDPs) have remained elusive. IDPs are abundant in eukaryotes and found to associate with NPs intracellularly. As a model system, we focused on ultrasmall gold nanoparticles (usGNPs) and tau, a cytosolic IDP associated with Alzheimer's disease. Using site-resolved NMR, steady-state fluorescence, calorimetry, and circular dichroism, we reveal that tau and usGNPs form stable multimolecular assemblies, representing a new type of nano-bio interaction. Specifically, the observed interaction hot spots explain the influence of usGNPs on tau conformational transitions, with implications for the intracellular targeting of aberrant IDP aggregation.
    Keywords:  NMR spectroscopy; intrinsically disordered proteins; protein aggregation; protein−nanoparticle interaction; ultrasmall nanoparticles
    DOI:  https://doi.org/10.1021/acs.nanolett.2c02902
  5. Methods Mol Biol. 2023 ;2552 125-139
      This chapter describes the application of constrained geometric simulations for prediction of antibody structural dynamics. We utilize constrained geometric simulations method FRODAN, which is a low computational complexity alternative to molecular dynamics (MD) simulations that can rapidly explore flexible motions in protein structures. FRODAN is highly suited for conformational dynamics analysis of large proteins, complexes, intrinsically disordered proteins, and dynamics that occurs on longer biologically relevant time scales that are normally inaccessible to classical MD simulations. This approach predicts protein dynamics at an all-atom scale while retaining realistic covalent bonding, maintaining dihedral angles in energetically good conformations while avoiding steric clashes in addition to performing other geometric and stereochemical criteria checks. In this chapter, we apply FRODAN to showcase its applicability for probing functionally relevant dynamics of IgG2a, including large-amplitude domain-domain motions and motions of complementarity determining region (CDR) loops. As was suggested in previous experimental studies, our simulations show that antibodies can explore a large range of conformational space.
    Keywords:  Antibody dynamics; Geometric simulations; Protein flexibility; Rigidity Theory
    DOI:  https://doi.org/10.1007/978-1-0716-2609-2_6
  6. Biomolecules. 2022 Oct 26. pii: 1566. [Epub ahead of print]12(11):
      Intrinsically disordered proteins (IDPs) lack well-defined 3D structures and can only be described as ensembles of different conformations. This high degree of flexibility allows them to interact promiscuously and makes them capable of fulfilling unique and versatile regulatory roles in cellular processes. These functional benefits make IDPs widespread in nature, existing in every living organism from bacteria and fungi to plants and animals. Due to their open and exposed structural state, IDPs are much more prone to proteolytic degradation than their globular counterparts. Therefore, the purification of recombinant IDPs requires extra care and caution, such as maintaining low temperature throughout the purification, the use of protease inhibitor cocktails and fast workflow. Even so, in the case of long IDP targets, the appearance of truncated by-products often seems unavoidable. The separation of these unwanted proteins can be very challenging due to their similarity to the parent target protein. Here, we describe a tandem-tag purification method that offers a remedy to this problem. It contains only common affinity-chromatography steps (HisTrap and Heparin) to ensure low cost, easy access and scaling-up for possible industrial use. The effectiveness of the method is demonstrated with four examples, Tau-441 and two of its fragments and the transactivation domain (AF1) of androgen receptor.
    Keywords:  Tau; affinity chromatography; androgen receptor (AF1); intrinsically disordered proteins (IDPs); protein purification
    DOI:  https://doi.org/10.3390/biom12111566
  7. Front Mol Biosci. 2022 ;9 1021939
      Phase separation of intrinsically disordered proteins (IDPs) is a phenomenon associated with many essential cellular processes, but a robust method to compute the binodal from molecular dynamics simulations of IDPs modeled at the all-atom level in explicit solvent is still elusive, due to the difficulty in preparing a suitable initial dense configuration and in achieving phase equilibration. Here we present SpiDec as such a method, based on spontaneous phase separation via spinodal decomposition that produces a dense slab when the system is initiated at a homogeneous, low density. After illustrating the method on four model systems, we apply SpiDec to a tetrapeptide modeled at the all-atom level and solvated in TIP3P water. The concentrations in the dense and dilute phases agree qualitatively with experimental results and point to binodals as a sensitive property for force-field parameterization. SpiDec may prove useful for the accurate determination of the phase equilibrium of IDPs.
    Keywords:  binodal; biomolecular condensates; interfacial tension; phase equilibrium; phase separation; spinodal decomposition
    DOI:  https://doi.org/10.3389/fmolb.2022.1021939
  8. Essays Biochem. 2022 Nov 09. pii: EBC20220060. [Epub ahead of print]
      Phosphorylation is the most common post-translational modification (PTM) in eukaryotes, occurring particularly frequently in intrinsically disordered proteins (IDPs). These proteins are highly flexible and dynamic by nature. Thus, it is intriguing that the addition of a single phosphoryl group to a disordered chain can impact its function so dramatically. Furthermore, as many IDPs carry multiple phosphorylation sites, the number of possible states increases, enabling larger complexities and novel mechanisms. Although a chemically simple and well-understood process, the impact of phosphorylation on the conformational ensemble and molecular function of IDPs, not to mention biological output, is highly complex and diverse. Since the discovery of the first phosphorylation site in proteins 75 years ago, we have come to a much better understanding of how this PTM works, but with the diversity of IDPs and their capacity for carrying multiple phosphoryl groups, the complexity grows. In this Essay, we highlight some of the basic effects of IDP phosphorylation, allowing it to serve as starting point when embarking on studies into this topic. We further describe how recent complex cases of multisite phosphorylation of IDPs have been instrumental in widening our view on the effect of protein phosphorylation. Finally, we put forward perspectives on the phosphorylation of IDPs, both in relation to disease and in context of other PTMs; areas where deep insight remains to be uncovered.
    Keywords:  allosteric regulation; intrinsically disordered proteins; molecular mechanisms; phosphorylation/dephosphorylation; post translational modification; protein-protein interactions
    DOI:  https://doi.org/10.1042/EBC20220060
  9. J Mol Biol. 2022 Nov 08. pii: S0022-2836(22)00498-3. [Epub ahead of print] 167878
      Bacterial functional amyloids contribute to biofilm development by bacteria and provide protection from the immune system and prevent antibiotic treatment. Strategies to target amyloid formation and interrupt biofilm formation have attracted recent interest due to their antimicrobial potential. Functional amyloid in Pseudomonas (Fap) includes FapC as the major component of the fibril while FapB is a minor component suggested to function as a nucleator of FapC. The system also includes the small periplasmic protein FapA, which has been shown to regulate fibril composition and morphology. The interplay between these three components is central in Fap fibril biogenesis. Here we present a comprehensive biophysical and spectroscopy analysis of FapA, FapB and FapC and provide insight into their molecular interactions. We show that all three proteins are primarily disordered with some regions with structural propensities for α-helix and β-sheet. FapA inhibits FapC fibrillation by targeting the nucleation step, whereas for FapB the elongation step is modulated. Furthermore, FapA alters the morphology of FapC (more than FapB) fibrils. Complex formation is observed between FapA and FapC, but not between FapA and FapB, and likely involves the N-terminus of FapA. We conclude that FapA is an intrinsically disordered chaperone for FapC that guards against fibrillation within the periplasm. This new understanding of a natural protective mechanism of Pseudomonas against amyloid formations can serve as inspiration for strategies blocking biofilm formation in infections.
    Keywords:  FIDA; Intrinsically disordered protein; NMR; SAXS; amyloidogenesis; inhibitor
    DOI:  https://doi.org/10.1016/j.jmb.2022.167878
  10. Acc Chem Res. 2022 Nov 08.
      ConspectusProtein folding and dynamics are controlled by an interplay of thermal and viscosity effects. The effect of viscous drag through the solvent molecules is described by the classic Kramers theory in the high friction limit, which considers the dampening of the reactant molecules in the solution and quantifies the dependence of the reaction rate on the frictional drag. In addition to the external energy dissipation originating from the surrounding solvent molecules, there is an additional mode of internal energy dissipative force operative within the polypeptide chain reflecting the internal resistance of the chain to its conformational alterations. This dry, solvent-independent intrinsic frictional drag is termed internal friction. In the case of natively folded proteins, the physical origin of internal friction is primarily attributed to the intrachain interactions or other nonnative interactions in their compact states. However, the molecular origin of internal friction in intrinsically disordered proteins (IDPs) remains elusive.In this Account, we address this fundamental issue: what are the principal drivers of viscosity-independent (dry) friction in highly solvated, expanded, conformationally flexible, rapidly fluctuating IDPs that do not possess persistent intrachain interactions? IDPs exhibit diffusive conformational dynamics that is predominantly dominated by the segmental motion of the backbone arising due to the dihedral rotations in the Ramachandran Φ-Ψ space. The physical origin of friction in a complex biopolymeric system such as IDPs can be described by classic polymer models, namely, Rouse/Zimm models with internal friction. These one-dimensional models do not invoke torsional fluctuation components. Kuhn's classic description includes the connection between internal friction and microscopic dihedral hopping. Based on our time-resolved fluorescence anisotropy results, we describe that the sequence-dependent, collective, short-range backbone dihedral rotations govern localized internal friction in an archetypal IDP, namely, α-synuclein. The highly sensitive, residue-specific fluorescence depolarization kinetics offers a potent methodology to characterize and quantify the directional decorrelation engendered due to the short-range dihedral relaxation of the polypeptide backbone in the dihedral space. We utilized this characteristic relaxation time scale as our dynamic readout to quantify the site-specific frictional component. Our linear viscosity-dependent model of torsional relaxation time scale furnished a finite nonzero time constant at the zero solvent viscosity representing the solvent-independent internal friction. These results unveil the effect of the degree of dihedral restraining parameter on the internal friction component by showing that a restrained proline residue imparts higher torsional stiffness in the chain segments and, therefore, exhibits higher internal friction. This Account sheds light on the molecular underpinning of the sequence-specific internal friction in IDPs and will be of interest to unmask the role of internal friction in a diverse range of biomolecular processes involving binding-induced folding, allosteric interaction, protein misfolding and aggregation, and biomolecular condensation via phase separation.
    DOI:  https://doi.org/10.1021/acs.accounts.2c00528
  11. J Phys Chem B. 2022 Nov 07.
      A theory for sequence-dependent liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) in the study of biomolecular condensates is formulated by extending the random phase approximation (RPA) and field-theoretic simulation (FTS) of heteropolymers with spatially long-range Coulomb interactions to include the fundamental effects of short-range, hydrophobic-like interactions between amino acid residues. To this end, short-range effects are modeled by Yukawa interactions between multiple nonelectrostatic charges derived from an eigenvalue decomposition of pairwise residue-residue contact energies. Chain excluded volume is afforded by incompressibility constraints. A mean-field approximation leads to an effective Flory-Huggins χ parameter, which, in conjunction with RPA, accounts for the contact-interaction effects of amino acid composition and the sequence-pattern effects of long-range electrostatics in IDP LLPS, whereas FTS based on the formulation provides full sequence dependence for both short- and long-range interactions. This general approach is illustrated here by applications to variants of a natural IDP in the context of several different amino-acid interaction schemes as well as a set of different model hydrophobic-polar sequences sharing the same composition. Effectiveness of the methodology is verified by coarse-grained explicit-chain molecular dynamics simulations.
    DOI:  https://doi.org/10.1021/acs.jpcb.2c06181
  12. Methods. 2022 Nov 02. pii: S1046-2023(22)00232-8. [Epub ahead of print]208 59-65
      RNA-binding proteins (RBPs) typically bind to RNA in a sequence-specific manner, resulting in post-transcriptional gene regulation. While the various classes of RNA-binding domains are largely structured, flexible linkers are frequently observed between them. Emerging evidence suggests that these unstructured regions may help spatially position the RNA-binding domains allowing for RNA binding and/or may contribute directly to RNA association via certain sequence motifs contained within them. The importance of these unstructured regions is widely appreciated; however, understanding their contribution to RNA binding, protein stability, and function has been difficult to ascertain. Thus, it is crucial to have a set of rapid and economical assays that do not require specialized instrumentation to study their impact on RBP function. Herein, we discuss the use of plate-based and cell-based thermal shift assays to study the impact of the intrinsically disordered region on the function of a highly conserved RBP, hnRNP K.
    Keywords:  CETSA; Intrinsic disorder; Poly-C binding proteins; Protein thermal shift; hnRNP K
    DOI:  https://doi.org/10.1016/j.ymeth.2022.10.009
  13. J Cell Biol. 2022 Dec 05. pii: e202205069. [Epub ahead of print]221(12):
      The amplitude of Wnt/β-catenin signaling is precisely controlled by the assembly of the cell surface-localized Wnt receptor signalosome and the cytosolic β-catenin destruction complex. How these two distinct complexes are coordinately controlled remains largely unknown. Here, we demonstrated that the signalosome scaffold protein Dishevelled 2 (Dvl2) undergoes liquid-liquid phase separation (LLPS). Dvl2 LLPS is mediated by an intrinsically disordered region and facilitated by components of the signalosome, such as the receptor Fzd5. Assembly of the signalosome is initiated by rapid recruitment of Dvl2 to the membrane, followed by slow and dynamic recruitment of Axin1. Axin LLPS mediates assembly of the β-catenin destruction complex, and Dvl2 attenuates LLPS of Axin. Compared with the destruction complex, Axin partitions into the signalosome at a lower concentration and exhibits a higher mobility. Together, our results revealed that Dvl2 LLPS is crucial for controlling the assembly of the Wnt receptor signalosome and disruption of the phase-separated β-catenin destruction complex.
    DOI:  https://doi.org/10.1083/jcb.202205069
  14. Chembiochem. 2022 Nov 06.
      The protein high mobility group A1 (HMGA1) is an important regulator of chromatin organization and function. However, the mechanisms by which it exerts its biological function are not fully understood. Here, we report that the HMGA isoform, HMGA1a, nucleates into foci that display liquid-like properties in the nucleus, and that the protein readily undergoes phase separation to form liquid condensates in vitro. By bringing together machine-leaning modelling, cellular and biophysical experiments and multiscale simulations, we demonstrate that phase separation of HMGA1a is critically promoted by protein-DNA interactions, and has the potential to be modulated by post-transcriptional effects such as phosphorylation. We further show that the intrinsically disordered C-terminal tail of HMGA1a significantly contributes to its phase separation through cation-π and electrostatic interactions. Our work sheds light on HMGA1 phase separation as an emergent biophysical factor in regulating chromatin structure.
    Keywords:  Chromatin regulator; HMGA; Protein-DNA interactions; liquid-liquid phase separation; phase diagram
    DOI:  https://doi.org/10.1002/cbic.202200450
  15. Biomolecules. 2022 Oct 27. pii: 1573. [Epub ahead of print]12(11):
      (1) Background: Prion-like transcellular spreading of tau pathology in Alzheimer's disease (AD) is mediated by tau binding to the cell-surface glycan heparan sulfate (HS). However, the structural determinants for tau-HS interaction are not well understood. (2) Methods and Results: Binding-site mapping using NMR showed two major binding regions in full-length tau responsible for heparin interaction. Thus, two tau constructs, tau PRR2* and tau R2*, were designed to investigate the molecular details at the tau-heparin binding interface. The 2D 1H-15N HSQC of tau PRR2* and tau R2* lacked dispersion, which is characteristic for intrinsically disordered proteins. NMR titration of Arixtra into 15N-labeled tau R2* induced large chemical shift perturbations (CSPs) in 275VQIINK280 and downstream residues K281-D283, in which L282 and I278 displayed the largest shifts. NMR titration of Arixtra into 15N-labeled tau PRR2* induced the largest CSPs for residue R209 followed by residues S210 and R211. Residue-based CSP fitting showed that tau PRR2*-Arixtra interaction had a much stronger binding affinity (0.37-0.67 mM) than that of tau R2*-Arixtra (1.90-5.12 mM) interaction. (3) Conclusions: Our results suggested that PRR2 is a crucial domain for tau-heparin and tau-HS interaction.
    Keywords:  Alzheimer’s disease; heparin; proline-rich region; tau
    DOI:  https://doi.org/10.3390/biom12111573
  16. Biochemistry. 2022 Nov 07.
      Various neurological dysfunctions are associated with cytotoxic amyloid-containing aggregates formed through the irreversible maturation of protein condensates generated by phase separation. Here, we investigate the amino acid code for this cytotoxicity using TDP-43 deep-sequencing data. Within the droplet landscape framework, we analyze the impact of mutations in the amyloid core, aggregation hot-spot, and droplet-promoting residues on TDP-43 cytotoxicity. Our analysis suggests that TDP-43 mutations associated with low cytotoxicity moderately decrease the probability of droplet formation while increasing the probability of multimodal binding. These mutations promote both ordered and disordered binding modes, thus facilitating the conversion between the droplet and amyloid states. Based on this understanding, we develop an extension of the FuzDrop method for the sequence-based prediction of the cytotoxicity of aging condensates and test it over 20,000 TDP-43 variants. Our analysis provides insight into the amino acid code that regulates the cytotoxicity associated with the maturation of liquid-like condensates into amyloid-containing aggregates, suggesting that, at least in the case of TDP-43, mutations that promote aggregation tend to decrease cytotoxicity, while those that promote droplet formation tend to increase cytotoxicity.
    DOI:  https://doi.org/10.1021/acs.biochem.2c00499