bims-indpro Biomed News
on Intrinsically disordered proteins
Issue of 2022–07–03
thirteen papers selected by
Sara Mingu, Johannes Gutenberg University
  1. The free energy folding penalty accompanying binding of intrinsically disordered α-helical motifs.
  2. Investigating Intrinsically Disordered Proteins With Brownian Dynamics.
  3. Multivalent Interaction of Beta-Catenin With its Intrinsically Disordered Binding Partner Adenomatous Polyposis Coli.
  4. More is simpler: Decomposition of ligand-binding affinity for proteins being disordered.
  5. Intrinsically Disordered Protein Condensate-Modified Surface for Mitigation of Biofouling and Foreign Body Response.
  6. Discovering molecular features of intrinsically disordered regions by using evolution for contrastive learning.
  7. Quantitative Frameworks for Multivalent Macromolecular Interactions in Biological Linear Lattice Systems.
  8. Extended disordered regions of ribosome-associated NAC proteins paralogs belong only to the germline in Drosophila melanogaster.
  9. A review of the effects of ATP and hydroxychloroquine on the phase separation of the SARS-CoV-2 nucleocapsid protein.
  10. Lsm7 phase-separated condensates trigger stress granule formation.
  11. Impact of Electronic Polarization on Preformed, β-Strand Rich Homogenous and Heterogenous Amyloid Oligomers.
  12. Activation of p53: how phosphorylated Ser15 triggers sequential phosphorylation of p53 at Thr18 by CK1δ.
  13. Targeting Protein Interaction Hotspots Using Structured and Disordered Chimeric Peptide Inhibitors.
  1. Protein Sci. 2022 Jul;31(7): e4370
    The free energy folding penalty accompanying binding of intrinsically disordered α-helical motifs.
    San Hadži, Jurij Lah.
      Intrinsically disordered proteins (IDPs) are abundant in eukaryotic proteomes and preform critical roles in many cellular processes, most often through the association with globular proteins. Despite lacking a stable three-dimensional structure by themselves, they may acquire a defined conformation upon binding globular targets. The most common type of secondary structure acquired by these binding motifs entails formation of an α-helix. It has been hypothesized that such disorder-to-order transitions are associated with a significant free energy penalty due to IDP folding, which reduces the overall IDP-target affinity. However, the exact magnitude of IDP folding penalty in α-helical binding motifs has not been systematically estimated. Here, we report the folding penalty contributions for 30 IDPs undergoing folding-upon-binding and find that the average IDP folding penalty is +2.0 kcal/mol and ranges from 0.7 to 3.5 kcal/mol. We observe that the folding penalty scales approximately linearly with the change in IDP helicity upon binding, which provides a simple empirical way to estimate folding penalty. We analyze to what extent do pre-structuring and target-bound IDP dynamics (fuzziness) reduce the folding penalty and find that these effects combined, on average, reduce the folding cost by around half. Taken together, the presented analysis provides a quantitative basis for understanding the role of folding penalty in IDP-target interactions and introduces a method estimate this quantity. Estimation and reduction of IDP folding penalty may prove useful in the rational design of helix-stabilized inhibitors of IDP-target interactions. STATEMENT: The α-helical binding motifs are ubiquitous among the intrinsically disordered proteins (IDPs). Upon binding their targets, they undergo a disorder-to-order transition, which is accompanied by a significant folding penalty whose magnitude is generally not known. Here, we use recently developed statistical-thermodynamic model to estimate the folding penalties for 30 IDPs and clarify the roles of IDP pre-folding and bound-state dynamics in reducing the folding penalty.
    Keywords:  binding motif; folding penalty; folding-upon-binding; fuzziness; intrinsically disordered proteins; peptide inhibitor; pre-folding
    DOI:  https://doi.org/10.1002/pro.4370
  2. Front Mol Biosci. 2022 ;9 898838
    Investigating Intrinsically Disordered Proteins With Brownian Dynamics.
    Surl-Hee Ahn, Gary A Huber, J Andrew McCammon.
      Intrinsically disordered proteins (IDPs) have recently become systems of great interest due to their involvement in modulating many biological processes and their aggregation being implicated in many diseases. Since IDPs do not have a stable, folded structure, however, they cannot be easily studied with experimental techniques. Hence, conducting a computational study of these systems can be helpful and be complementary with experimental work to elucidate their mechanisms. Thus, we have implemented the coarse-grained force field for proteins (COFFDROP) in Browndye 2.0 to study IDPs using Brownian dynamics (BD) simulations, which are often used to study large-scale motions with longer time scales and diffusion-limited molecular associations. Specifically, we have checked our COFFDROP implementation with eight naturally occurring IDPs and have investigated five (Glu-Lys)25 IDP sequence variants. From measuring the hydrodynamic radii of eight naturally occurring IDPs, we found the ideal scaling factor of 0.786 for non-bonded interactions. We have also measured the entanglement indices (average C α distances to the other chain) between two (Glu-Lys)25 IDP sequence variants, a property related to molecular association. We found that entanglement indices decrease for all possible pairs at excess salt concentration, which is consistent with long-range interactions of these IDP sequence variants getting weaker at increasing salt concentration.
    Keywords:  Browndye; Brownian dynamics simulation; COFFDROP force field; intrinsically disordered proteins; molecular associations
    DOI:  https://doi.org/10.3389/fmolb.2022.898838
  3. Front Mol Biosci. 2022 ;9 896493
    Multivalent Interaction of Beta-Catenin With its Intrinsically Disordered Binding Partner Adenomatous Polyposis Coli.
    Pamela J E Rowling, Ben L Murton, Zhen Du, Laura S Itzhaki.
      The Wnt signalling pathway plays key roles in cell proliferation, differentiation and fate decisions in embryonic development and maintenance of adult tissues, and the twelve Armadillo (ARM) repeat-containing protein β-catenin acts as the signal transducer in this pathway. Here we investigate the interaction between β-catenin's ARM repeat domain and the intrinsically disordered protein adenomatous polyposis coli (APC). APC is a giant multivalent scaffold that brings together the different components of the so-called "β-catenin destruction complex", which drives β-catenin degradation via the ubiquitin-proteasome pathway. Mutations and truncations in APC, resulting in loss of APC function and hence elevated β-catenin levels and upregulation of Wnt signalling, are associated with numerous cancers including colorectal carcinomas. APC has a long intrinsically disordered region (IDR) that contains a series of 15-residue and 20-residue binding regions for β-catenin. Here we explore the multivalent nature of the interaction of β-catenin with the highest affinity APC repeat, both at equilibrium and under kinetic conditions. We use a combination of single-site substitutions, deletions and insertions to dissect the mechanism of molecular recognition and the roles of the three β-catenin-binding subdomains of APC.
    Keywords:  adenomatous polyposis coli (APC); armadillo repeat; beta-catenin (β-catenin); fuzzy binding; intrinsically disordered protein; multivalency; protein-protein interaction (PPI)
    DOI:  https://doi.org/10.3389/fmolb.2022.896493
  4. Protein Sci. 2022 Jul;31(7): e4375
    More is simpler: Decomposition of ligand-binding affinity for proteins being disordered.
    Xiaohui Wang, Bin Chong, Zhaoxi Sun, Hao Ruan, Yingguang Yang, Pengbo Song, Zhirong Liu.
      In statistical mechanics, it is well known that the huge number of degrees of freedom does not complicate the problem as it seems, but actually greatly simplifies the analysis (e.g., to give a Boltzmann distribution). Here, we reveal that the ensemble averaging from the vast conformations of intrinsically disordered proteins (IDPs) greatly simplifies the nature of binding affinity, which can be reliably decomposed into a sum of the ligandability of IDP and the capacity of ligand. Such an unexpected regularity is applied to facilitate the virtual screening upon IDPs. It also provides essential insight in understanding the specificity difference between IDPs and conventional ordered proteins since the specificity is caused by deviation from the baseline behavior of protein-ligand binding.
    Keywords:  affinity; druggability; druglikeness; intrinsically disordered proteins; leadlikeness; ligandability; molecular docking; specificity; virtual screening
    DOI:  https://doi.org/10.1002/pro.4375
  5. J Am Chem Soc. 2022 Jun 29.
    Intrinsically Disordered Protein Condensate-Modified Surface for Mitigation of Biofouling and Foreign Body Response.
    Rong Chang, Jia-Lin Chen, Guan-Yi Zhang, Yue Li, Hua-Zhen Duan, Shi-Zhong Luo, Yong-Xiang Chen.
      Mitigation of biofouling and the host's foreign body response (FBR) is a critical challenge with biomedical implants. The surface coating with various anti-fouling materials provides a solution to overcome it, but limited options in clinic and their potential immunogenicity drive the development of more alternative coating materials. Herein, inspired by liquid-liquid phase separation of intrinsically disordered proteins (IDPs) to form separated condensates in physiological conditions, we develop a new type of low-fouling biomaterial based on flexible IDP of FUS protein containing rich hydrophilic residues. A chemical structure-defined FUS IDP sequence tagged with a tetra-cysteine motif (IDPFUS) was engineered and applied for covalent immobilization on various surfaces to form a uniform layer of protein tangles, which boosted strong hydration on surfaces, as revealed by molecular dynamics simulation. The IDPFUS-coated surfaces displayed excellent performance in resisting adsorption of various proteins and adhesion of different cells, platelets, and bacteria. Moreover, the IDPFUS-coated implants largely mitigated the host's FBR compared with bare implants and particularly outperformed PEG-coated implants in reducing collagen encapsulation. Thus, this novel low-fouling and anti-FBR strategy provides a potential surface coating material for biomedical implants, which will also shed light on exploring similar applications of other IDP proteins.
    DOI:  https://doi.org/10.1021/jacs.2c02677
  6. PLoS Comput Biol. 2022 Jun 29. 18(6): e1010238
    Discovering molecular features of intrinsically disordered regions by using evolution for contrastive learning.
    Alex X Lu, Amy X Lu, Iva Pritišanac, Taraneh Zarin, Julie D Forman-Kay, Alan M Moses.
      A major challenge to the characterization of intrinsically disordered regions (IDRs), which are widespread in the proteome, but relatively poorly understood, is the identification of molecular features that mediate functions of these regions, such as short motifs, amino acid repeats and physicochemical properties. Here, we introduce a proteome-scale feature discovery approach for IDRs. Our approach, which we call "reverse homology", exploits the principle that important functional features are conserved over evolution. We use this as a contrastive learning signal for deep learning: given a set of homologous IDRs, the neural network has to correctly choose a held-out homolog from another set of IDRs sampled randomly from the proteome. We pair reverse homology with a simple architecture and standard interpretation techniques, and show that the network learns conserved features of IDRs that can be interpreted as motifs, repeats, or bulk features like charge or amino acid propensities. We also show that our model can be used to produce visualizations of what residues and regions are most important to IDR function, generating hypotheses for uncharacterized IDRs. Our results suggest that feature discovery using unsupervised neural networks is a promising avenue to gain systematic insight into poorly understood protein sequences.
    DOI:  https://doi.org/10.1371/journal.pcbi.1010238
  7. Mol Cells. 2022 Jun 27.
    Quantitative Frameworks for Multivalent Macromolecular Interactions in Biological Linear Lattice Systems.
    Jaejun Choi, Ryeonghyeon Kim, Junseock Koh.
      Multivalent macromolecular interactions underlie dynamic regulation of diverse biological processes in ever-changing cellular states. These interactions often involve binding of multiple proteins to a linear lattice including intrinsically disordered proteins and the chromosomal DNA with many repeating recognition motifs. Quantitative understanding of such multivalent interactions on a linear lattice is crucial for exploring their unique regulatory potentials in the cellular processes. In this review, the distinctive molecular features of the linear lattice system are first discussed with a particular focus on the overlapping nature of potential protein binding sites within a lattice. Then, we introduce two general quantitative frameworks, combinatorial and conditional probability models, dealing with the overlap problem and relating the binding parameters to the experimentally measurable properties of the linear lattice-protein interactions. To this end, we present two specific examples where the quantitative models have been applied and further extended to provide biological insights into specific cellular processes. In the first case, the conditional probability model was extended to highlight the significant impact of nonspecific binding of transcription factors to the chromosomal DNA on gene-specific transcriptional activities. The second case presents the recently developed combinatorial models to unravel the complex organization of target protein binding sites within an intrinsically disordered region (IDR) of a nucleoporin. In particular, these models have suggested a unique function of IDRs as a molecular switch coupling distinct cellular processes. The quantitative models reviewed here are envisioned to further advance for dissection and functional studies of more complex systems including phase-separated biomolecular condensates.
    Keywords:  biological linear lattice; combinatorial model; conditional probability model; multivalent binding; overlapping binding site
    DOI:  https://doi.org/10.14348/molcells.2022.0035
  8. Sci Rep. 2022 Jul 01. 12(1): 11191
    Extended disordered regions of ribosome-associated NAC proteins paralogs belong only to the germline in Drosophila melanogaster.
    Galina L Kogan, Elena A Mikhaleva, Oxana M Olenkina, Sergei S Ryazansky, Oxana V Galzitskaya, Yuri A Abramov, Toomas A Leinsoo, Natalia V Akulenko, Sergey A Lavrov, Vladimir A Gvozdev.
      The nascent polypeptide-associated complex (NAC) consisting of α- and β-subunits is an essential ribosome-associated protein conserved in eukaryotes. NAC is a ubiquitously expressed co-translational regulator of nascent protein folding and sorting providing for homeostasis of cellular proteins. Here we report on discovering the germline-specific NACαβ paralogs (gNACs), whose β-subunits, non-distinguishable by ordinary immunodetection, are encoded by five highly homologous gene copies, while the α-subunit is encoded by a single αNAC gene. The gNAC expression is detected in the primordial embryonic and adult gonads via immunostaining. The germline-specific α and β subunits differ from the ubiquitously expressed paralogs by the extended intrinsically disordered regions (IDRs) acquired at the N- and C-termini of the coding regions, predicted to be phosphorylated. The presence of distinct phosphorylated isoforms of gNAC-β subunits is confirmed by comparing of their profiles by 2D-isoeletrofocusing resolution before and after phosphatase treatment of testis ribosomes. We revealed that the predicted S/T sites of phosphorylation in the individual orthologous IDRs of gNAC-β sequences of Drosophila species are positionally conserved despite these disordered regions are drastically different. We propose the IDR-dependent molecular crowding and specific coordination of NAC and other proteostasis regulatory factors at the ribosomes of germinal cells. Our findings imply that there may be a functional crosstalk between the germinal and ubiquitous α- and β-subunits based on assessing their depletion effects on the fly viability and gonad development.
    DOI:  https://doi.org/10.1038/s41598-022-15233-3
  9. Biophys Rev. 2022 Jun 22. 1-7
    A review of the effects of ATP and hydroxychloroquine on the phase separation of the SARS-CoV-2 nucleocapsid protein.
    Mei Dang, Jianxing Song.
      SARS-CoV-2 is the coronavirus causing the ongoing pandemic with > 460 millions of infections and > 6 millions of deaths. SARS-CoV-2 nucleocapsid (N) is the only structural protein which plays essential roles in almost all key steps of the viral life cycle with its diverse functions depending on liquid-liquid phase separation (LLPS) driven by interacting with various nucleic acids. The 419-residue N protein is highly conserved in all variants including delta and omicron, and composed of both folded N-/C-terminal domains (NTD/CTD) as well as three long intrinsically disordered regions (IDRs). Recent results have suggested that its CTD and IDRs are also cryptic nucleic acid-binding domains. In this context, any small molecules capable of interfering in its interaction with nucleic acids are anticipated to modulate its LLPS and associated functions. Indeed, ATP, the energy currency existing at very high concentrations (2-12 mM) in all living cells but absent in viruses, modulates LLPS of N protein, and consequently appears to be evolutionarily hijacked by SARS-CoV-2 to promote its life cycle. Hydroxychloroquine (HCQ) has been also shown to specifically bind NTD and CTD to inhibit their interactions with nucleic acids, as well as to disrupt LLPS. Particularly, the unique structure of the HCQ-CTD complex offers a promising strategy for further design of anti-SARS-CoV-2 drugs with better affinity and specificity. The finding may indicate that LLPS is indeed druggable by small molecules, thus opening up a promising direction for drug discovery/design by targeting LLPS in general.
    Keywords:  Adenosine triphosphate (ATP); Hydroxychloroquine (HCQ): SARS-CoV-2; Liquid–liquid phase separation (LLPS); NMR spectroscopy; Nucleocapsid (N) protein
    DOI:  https://doi.org/10.1007/s12551-022-00957-3
  10. Nat Commun. 2022 Jun 28. 13(1): 3701
    Lsm7 phase-separated condensates trigger stress granule formation.
    Michelle Lindström, Lihua Chen, Shan Jiang, Dan Zhang, Yuan Gao, Ju Zheng, Xinxin Hao, Xiaoxue Yang, Arpitha Kabbinale, Johannes Thoma, Lisa C Metzger, Deyuan Y Zhang, Xuefeng Zhu, Huisheng Liu, Claes M Gustafsson, Björn M Burmann, Joris Winderickx, Per Sunnerhagen, Beidong Liu.
      Stress granules (SGs) are non-membranous organelles facilitating stress responses and linking the pathology of age-related diseases. In a genome-wide imaging-based phenomic screen, we identify Pab1 co-localizing proteins under 2-deoxy-D-glucose (2-DG) induced stress in Saccharomyces cerevisiae. We find that deletion of one of the Pab1 co-localizing proteins, Lsm7, leads to a significant decrease in SG formation. Under 2-DG stress, Lsm7 rapidly forms foci that assist in SG formation. The Lsm7 foci form via liquid-liquid phase separation, and the intrinsically disordered region and the hydrophobic clusters within the Lsm7 sequence are the internal driving forces in promoting Lsm7 phase separation. The dynamic Lsm7 phase-separated condensates appear to work as seeding scaffolds, promoting Pab1 demixing and subsequent SG initiation, seemingly mediated by RNA interactions. The SG initiation mechanism, via Lsm7 phase separation, identified in this work provides valuable clues for understanding the mechanisms underlying SG formation and SG-associated human diseases.
    DOI:  https://doi.org/10.1038/s41467-022-31282-8
  11. J Comput Biophys Chem. 2022 Jun;21(4): 449-460
    Impact of Electronic Polarization on Preformed, β-Strand Rich Homogenous and Heterogenous Amyloid Oligomers.
    Kelsie M King, Amanda K Sharp, Darcy S Davidson, Anne M Brown, Justin A Lemkul.
      Amyloids are a subset of intrinsically disordered proteins (IDPs) that self-assemble into cross-β oligomers and fibrils. The structural plasticity of amyloids leads to sampling of metastable, low-molecular-weight oligomers that contribute to cytotoxicity. Of interest are amyloid-β (Aβ) and islet amyloid polypeptide (IAPP), which are involved in the pathology of Alzheimer's disease and Type 2 Diabetes Mellitus, respectively. In addition to forming homogenous oligomers and fibrils, these species have been found to cross-aggregate in heterogeneous structures. Biophysical properties, including electronic effects, that are unique or conserved between homogenous and heterogenous amyloids oligomers are thus far unexplored. Here, we simulated homogenous and heterogenous amyloid oligomers of Aβ16-22 and IAPP20-29 fragments using the Drude oscillator model to investigate the impact of electronic polarization on the structural morphology and stability of preformed hexamers. Upon simulation of preformed, β-strand rich oligomers with Drude, structural rearrangement occurred causing some loss of β-strand structure in favor of random coil content for all oligomers. Homogenous Aβ16-22 was the most stable system, deriving stability from low polarization in hydrophobic residues and through salt bridge formation. Changes in polarization were observed primarily for Aβ16-22 residues in heterogenous cross-amyloid systems, displaying a decrease in charged residue dipole moments and an increase in hydrophobic sidechain dipole moments. This work is the first study utilizing the Drude-2019 force field with amyloid oligomers, providing insight into the impact of electronic effects on oligomer structure and highlighting the importance of different microenvironments on amyloid oligomer stability.
    Keywords:  Drude oscillator; amyloid-β; islet amyloid polypeptide (IAPP); polarizable molecular dynamics simulations
    DOI:  https://doi.org/10.1142/s2737416521420059
  12. Proteins. 2022 Jun 25.
    Activation of p53: how phosphorylated Ser15 triggers sequential phosphorylation of p53 at Thr18 by CK1δ.
    Sonia T Nicolaou, Srinivasaraghavan Kannan, Jim Warwicker, Chandra S Verma.
      The N-terminal transactivation domain (TAD) of p53 is a disordered region with multiple phosphorylation sites. Phosphorylation at Thr18 is crucial for the release of p53 from its negative regulator, MDM2. In stressed cells, CK1δ is responsible for phosphorylating Thr18, but requires Ser15 to be phosphorylated. To understand the mechanistic underpinnings of this sequential phosphorylation, molecular modelling and molecular dynamics simulation studies of these phosphorylation events were carried out. Our models suggest that a positively charged region on CK1δ near the ATP binding pocket, which is conserved across species, sequesters the negatively charged pSer15, thereby constraining the positioning of the rest of the peptide, such that the side chain of Thr18 is positioned close to the γ-phosphate of ATP. Furthermore, our studies show that the phosphorylated p53 TAD1 (p53pSer15) peptide binds more strongly to CK1δ than does p53. p53 adopts a helical structure when bound to CK1δ, which is lost upon phosphorylation at Ser15, thus gaining higher flexibility and ability to morph into the binding site. We propose that upon phosphorylation at Ser15 the p53 TAD1 peptide binds to CK1δ through an electrostatically driven induced fit mechanism resulting in a flanking fuzzy complex. This article is protected by copyright. All rights reserved.
    Keywords:  fuzzy complex; intrinsically disordered proteins; molecular dynamics; p53; phosphorylation
    DOI:  https://doi.org/10.1002/prot.26393
  13. ACS Chem Biol. 2022 Jun 27.
    Targeting Protein Interaction Hotspots Using Structured and Disordered Chimeric Peptide Inhibitors.
    Guy Mayer, Zohar Shpilt, Hadar Kowalski, Edit Y Tshuva, Assaf Friedler.
      The main challenge in inhibiting protein-protein interactions (PPI) for therapeutic purposes is designing molecules that bind specifically to the interaction hotspots. Adding to the complexity, such hotspots can be within both structured and disordered interaction interfaces. To address this, we present a strategy for inhibiting the structured and disordered hotspots of interactions using chimeric peptides that contain both structured and disordered parts. The chimeric peptides we developed are comprised of a cyclic structured part and a disordered part, which target both disordered and structured hotspots. We demonstrate our approach by developing peptide inhibitors for the interactions of the antiapoptotic iASPP protein. First, we developed a structured, α-helical stapled peptide inhibitor, derived from the N-terminal domain of MDM2. The peptide bound two hotspots on iASPP at the low micromolar range and had a cytotoxic effect on A2780 cancer cells with a half-maximal inhibitory concentration (IC50) value of 10 ± 1 μM. We then developed chimeric peptides comprising the structured stapled helical peptide and the disordered p53-derived LinkTer peptide that we previously showed to inhibit iASPP by targeting its disordered RT loop. The chimeric peptide targeted both structured and disordered domains in iASPP with higher affinity compared to the individual structured and disordered peptides and caused cancer cell death. Our strategy overcomes the inherent difficulty in inhibiting the interactions of proteins that possess structured and disordered regions. It does so by using chimeric peptides derived from different interaction partners that together target a much wider interface covering both the structured and disordered domains. This paves the way for developing such inhibitors for therapeutic purposes.
    DOI:  https://doi.org/10.1021/acschembio.2c00177
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