bims-indpro 2022-03-20 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022–03–20
fourteen papers selected by
Sara Mingu, Johannes Gutenberg University

Intrinsically disordered signaling proteins: Essential hub players in the control of stress responses in Saccharomyces cerevisiae.
Metadynamics Simulations to Study the Structural Ensembles and Binding Processes of Intrinsically Disordered Proteins.
The Role of ATP in Solubilizing RNA-binding Protein Fused in Sarcoma.
CsrA-Controlled Proteins Reveal New Dimensions of Acinetobacter baumannii Desiccation Tolerance.
Biophysics of endocytic vesicle formation: A focus on liquid-liquid phase separation.
An Intrinsically Disordered Peptide Tag that Confers an Unusual Solubility to Aggregation-Prone Proteins.
The PentUnFOLD algorithm as a tool to distinguish the dark and the light sides of the structural instability of proteins.
Proteomic analysis of heat-stable proteins revealed an increased proportion of proteins with compositionally biased regions.
The Cys-Pro motifs in the intrinsically disordered regions of the transcription factor BACH1 mediate distinct and overlapping functions upon heme binding.
Intrinsically disordered intracellular domains control key features of the mechanically-gated ion channel PIEZO2.
Peptide Dynamics and Metadynamics: Leveraging Enhanced Sampling Molecular Dynamics to Robustly Model Long-Timescale Transitions.
Dynamics of Amyloid Formation from Simplified Representation to Atomistic Simulations.
A "spindle and thread" mechanism unblocks p53 translation by modulating N-terminal disorder.
Kinetic interplay between droplet maturation and coalescence modulates shape of aged protein condensates.

PLoS One. 2022 ;17(3): e0265422

Intrinsically disordered signaling proteins: Essential hub players in the control of stress responses in Saccharomyces cerevisiae.

Leidys French-Pacheco, Omar Rosas-Bringas, Lorenzo Segovia, Alejandra A Covarrubias.

Cells have developed diverse mechanisms to monitor changes in their surroundings. This allows them to establish effective responses to cope with adverse environments. Some of these mechanisms have been well characterized in the budding yeast Saccharomyces cerevisiae, an excellent experimental model to explore and elucidate some of the strategies selected in eukaryotic organisms to adjust their growth and development in stressful conditions. The relevance of structural disorder in proteins and the impact on their functions has been uncovered for proteins participating in different processes. This is the case of some transcription factors (TFs) and other signaling hub proteins, where intrinsically disordered regions (IDRs) play a critical role in their function. In this work, we present a comprehensive bioinformatic analysis to evaluate the significance of structural disorder in those TFs (170) recognized in S. cerevisiae. Our findings show that 85.2% of these TFs contain at least one IDR, whereas ~30% exhibit a higher disorder level and thus were considered as intrinsically disordered proteins (IDPs). We also found that TFs contain a higher number of IDRs compared to the rest of the yeast proteins, and that intrinsically disordered TFs (IDTFs) have a higher number of protein-protein interactions than those with low structural disorder. The analysis of different stress response pathways showed a high content of structural disorder not only in TFs but also in other signaling proteins. The propensity of yeast proteome to undergo a liquid-liquid phase separation (LLPS) was also analyzed, showing that a significant proportion of IDTFs may undergo this phenomenon. Our analysis is a starting point for future research on the importance of structural disorder in yeast stress responses.

DOI: https://doi.org/10.1371/journal.pone.0265422
Methods Mol Biol. 2022 ;2405 169-178

Metadynamics Simulations to Study the Structural Ensembles and Binding Processes of Intrinsically Disordered Proteins.

Rui Zhou, Mojie Duan.

  The structures of intrinsically disordered proteins (IDPs) are highly dynamic. It is hard to characterize the structures of these proteins experimentally. Molecular dynamics (MD) simulation is a powerful tool in the understanding of protein dynamic structures and function. This chapter describes the application of metadynamics-based enhanced sampling methods in the study of phosphorylation regulation on the structure of kinase-inducible domains (KID). The structural properties of free pKID and KID were obtained by parallel tempering metadynamics combined with well-tempered ensemble (PTMetaD WTE) method, and the binding free energy surfaces of pKID/KID and KIX were characterized by bias-exchanged metadynamics (BE-MetaD) simulations.

Keywords:  Binding processes; Intrinsically disordered protein; Kinase-inducible domain; Metadynamics; Molecular dynamics simulations; Structure ensemble

DOI:  https://doi.org/10.1007/978-1-0716-1855-4_9
Proteins. 2022 Mar 16.

The Role of ATP in Solubilizing RNA-binding Protein Fused in Sarcoma.

Hayato Aida, Yasuteru Shigeta, Ryuhei Harada.

  Intrinsically disordered protein (IDP) plays an important role in liquid-liquid phase separation (LLPS). RNA-binding protein fused in sarcoma (FUS) is a well-studied IDP that induces LLPS since its low-complexity core region (FUS-LC-core) is essential for droplet formation through contacts between FUS-LC-cores. Several experimental studies have reported that adenosine triphosphate (ATP) concentrations modulate LLPS-driven droplet formation through the dissolution of FUS. To elucidate the role of ATP in this dissolution, microsecond-order all-atom molecular dynamics (MD) simulations were performed for a crowded system of FUS-LC-cores in the presence of multiple ATP molecules. Our analysis revealed that the adenine group of ATP frequently contacted the FUS-LC-core and the phosphoric acid group of ATP was exposed to the external solvent, which promoted both hydration and solubilization of FUS.

Keywords:  Adenosine triphosphate crowding; Dissolution; Intrinsically disordered protein; RNA-binding protein fused in sarcoma; Solubilization

DOI:  https://doi.org/10.1002/prot.26335
J Bacteriol. 2022 Mar 14. e0047921

CsrA-Controlled Proteins Reveal New Dimensions of Acinetobacter baumannii Desiccation Tolerance.

Yasuhiro Oda, Madelyn M Shapiro, Nathan M Lewis, Xuefei Zhong, Holly K Huse, Weizhi Zhong, James E Bruce, Colin Manoil, Caroline S Harwood.

  Hospital environments are excellent reservoirs for the opportunistic pathogen Acinetobacter baumannii in part because it is exceptionally tolerant to desiccation. We found that relative to other A. baumannii strains, the virulent strain AB5075 was strikingly desiccation resistant at 2% relative humidity (RH), suggesting that it is a good model for studies of the functional basis of this trait. Consistent with results from other A. baumannii strains at 40% RH, we found the global posttranscriptional regulator CsrA to be critically important for desiccation tolerance of AB5075 at 2% RH. Proteomics experiments identified proteins that were differentially present in wild-type and csrA mutant cells. Subsequent analysis of mutants in genes encoding some of these proteins revealed six genes that were required for wild-type levels of desiccation tolerance. These include genes for catalase, a universal stress protein, a hypothetical protein, and a biofilm-associated protein. Two genes of unknown function had very strong desiccation phenotypes, with one of the two genes predicting an intrinsically disordered protein (IDP) that binds to DNA. Intrinsically disordered proteins are widespread in eukaryotes but less so in prokaryotes. Our results suggest there are new mechanisms underlying desiccation tolerance in bacteria and identify several key functions involved. IMPORTANCE Acinetobacter baumannii is found in terrestrial environments but can cause nosocomial infections in very sick patients. A factor that contributes to the prevalence of A. baumannii in hospital settings is that it is intrinsically resistant to dry conditions. Here, we established the virulent strain A. baumannii AB5075 as a model for studies of desiccation tolerance at very low relative humidity. Our results show that this trait depends on two proteins of unknown function, one of which is predicted to be an intrinsically disordered protein. This category of protein is critical for the small animals named tardigrades to survive desiccation. Our results suggest that A. baumannii may have novel strategies to survive desiccation that have not previously been seen in bacteria.

Keywords:  Acinetobacter baumannii; CsrA; desiccation; intrinsically disordered proteins

DOI:  https://doi.org/10.1128/jb.00479-21
Curr Opin Cell Biol. 2022 Mar 10. pii: S0955-0674(22)00013-8. [Epub ahead of print]75 102068

Biophysics of endocytic vesicle formation: A focus on liquid-liquid phase separation.

Irene Schiano Lomoriello, Sara Sigismund, Kasey J Day.

Endocytosis is a fine-tuned mechanism of cellular communication through which cells internalize molecules on the plasma membrane, such as receptors and their bound ligands. Through receptor clustering in endocytic pits, recruitment of active receptors to different endocytic routes and their trafficking towards different fates, endocytosis modulates cell signaling and ultimately leads to a variety of biological responses. Many studies have focused their attention on specialized endocytic mechanisms depending on the nature of the internalizing cargo and cellular context, distinct sets of coat proteins, endocytic adaptors and membrane lipids. Here, we review recent advances in our understanding of the principles underlying endocytic vesicle formation, integrating both biochemical and biophysical factors, with a particular focus on intrinsically disordered regions (IDRs) creating weakly interconnected protein networks assembled through liquid-liquid phase separation (LLPS) and driving membrane bending especially in clathrin-mediated endocytosis (CME). We finally discuss how these properties impinge on receptor fate and signaling.

DOI: https://doi.org/10.1016/j.ceb.2022.02.002
Appl Environ Microbiol. 2022 Mar 14. e0009722

An Intrinsically Disordered Peptide Tag that Confers an Unusual Solubility to Aggregation-Prone Proteins.

Byung Hoon Jo.

  There is a high demand for the production of recombinant proteins in Escherichia coli for biotechnological applications, but their production is still limited by their insolubility. Fusion tags have been successfully used to enhance the solubility of aggregation-prone proteins; however, smaller and more powerful tags are desired for increasing the yield and quality of target proteins. Here, the NEXT tag, a 53-amino-acid-long solubility enhancer, is described. The NEXT tag showed outstanding ability to improve both in vivo and in vitro solubilities, with minimal effect on passenger proteins. The C-terminal region of the tag was mostly responsible for in vitro solubility, while the N-terminal region was essential for in vivo soluble expression. The NEXT tag appeared to be intrinsically disordered and seemed to exclude neighboring molecules and prevent protein aggregation by acting as an entropic bristle. This novel peptide tag should have general use as a fusion partner to increase the yield and quality of difficult-to-express proteins. IMPORTANCE Production of recombinant proteins in Escherichia coli still suffers from the insolubility problem. Conventional solubility enhancers with large sizes, represented by maltose-binding protein (MBP), have remained the first-choice tags; however, the success of the soluble expression of tagged proteins is largely unpredictable. In addition, the large tags can negatively affect the function of target proteins. In this work, the NEXT tag, an intrinsically disordered peptide, was introduced as a small but powerful alternative to MBP. The NEXT tag could significantly improve both the expression level and the solubility of target proteins, including a thermostable carbonic anhydrase and a polyethylene terephthalate (PET)-degrading enzyme that are remarkable enzymes for environmental bioremediation.

Keywords:  NEXT tag; PETase; carbonic anhydrase; entropic bristle; intrinsically disordered; soluble expression

DOI:  https://doi.org/10.1128/aem.00097-22
Amino Acids. 2022 Mar 16.

The PentUnFOLD algorithm as a tool to distinguish the dark and the light sides of the structural instability of proteins.

Victor Vitoldovich Poboinev, Vladislav Victorovich Khrustalev, Tatyana Aleksandrovna Khrustaleva, Tihon Evgenyevich Kasko, Vadim Dmitrievich Popkov.

  Intrinsically disordered proteins are frequently involved in important regulatory processes in the cell thanks to their ability to bind several different targets performing sometimes even opposite functions. The PentUnFOLD algorithm is a physicochemical method that is based on new propensity scales for disordered, nonstable and stable elements of secondary structure and on the counting of stabilizing and destabilizing intraprotein contacts. Unlike other methods, it works with a PDB file, and it can determine not only those fragments of alpha helices, beta strands, and random coils that can turn into disordered state (the "dark" side of the disorder), but also nonstable regions of alpha helices and beta strands which are able to turn into random coils (the "light" side), and vice versa (H ↔ C, E ↔ C). The scales have been obtained from structural data on disordered regions from the middle parts of amino acid sequences only, and not on their expectedly disordered N- and C-termini. Among other tendencies we have found that regions of both alpha helices and beta strands that can turn into the disordered state are relatively enriched in residues of Ala, Met, Asp, and Lys, while regions of both alpha helices and beta strands that can turn into random coil are relatively enriched in hydrophilic residues, and Cys, Pro, and Gly. Moreover, PentUnFOLD has the option to determine the effect of secondary structure transitions on the stability of a given region of a protein. The PentUnFOLD algorithm is freely available at http://3.17.12.213/pent-un-fold and http://chemres.bsmu.by/PentUnFOLD.htm .

Keywords:  Amino acid substitution; Computer algorithm; Human prion protein; Intrinsically disordered proteins; Structural shifts

DOI:  https://doi.org/10.1007/s00726-022-03153-5
Sci Rep. 2022 Mar 14. 12(1): 4347

Proteomic analysis of heat-stable proteins revealed an increased proportion of proteins with compositionally biased regions.

Hongsun Park, Tomoyuki Yamanaka, Nobuyuki Nukina.

Intrinsically disordered proteins (IDPs) have been in the spotlight for their unique properties, such as their lack of secondary structures and low sequence complexity. Alpha-synuclein and tau are representative disease-related IDPs with low complexity regions in their sequences, accumulating in the brains of patients with Parkinson disease and Alzheimer disease, respectively. Their heat resistance in particular was what attracted our attention. We assumed that there exist many other unidentified proteins that are resistant to heat-treatment, referred to as heat-stable proteins, which would also have low sequence complexity. In this study, we performed proteomic analysis of heat-stable proteins of mouse brains and found that proteins with compositionally biased regions are abundant in the heat-stable proteins. The proteins related to neurodegeneration are known to undergo different types of post-translational modifications (PTMs) such as phosphorylation and ubiquitination. We then investigated the heat-stability and aggregation properties of phosphorylated synuclein and tau with different phosphorylation sites. We suggest that PTMs can be important factors that determine the heat-stability and aggregation properties of a protein. IDPs identified in the heat-stable proteins of mouse brains would be candidates for the pathogenic proteins for neurodegeneration.

DOI: https://doi.org/10.1038/s41598-022-08044-z
FEBS Lett. 2022 Mar 18.

The Cys-Pro motifs in the intrinsically disordered regions of the transcription factor BACH1 mediate distinct and overlapping functions upon heme binding.

Kei Segawa, Kazuhiko Igarashi, Kazutaka Murayama.

  The function of the transcription factor BACH1 is regulated by heme binding to multiple Cys-Pro (CP) motifs within its intrinsically disordered regions. Here, biochemical analyses were conducted to reveal the individual functional roles of the CP motifs. Our findings revealed that four CP motifs in BACH1 individually contributed to the regulation of BACH1 activity by accepting heme in five- and six-coordination manner. The model structure around the bZip domain indicated that the CP motifs are in the N- and C-terminal heme-binding regions, which are approximately 9 nm apart, suggesting that spatial location is important for the individual function of the CP motifs. The presence of multiple CP motifs with distinct roles may ensure the multifaceted, strict regulation of BACH1 by heme.

Keywords:  CP motif; DNA binding; conformational flexibility; heme; protein-protein interaction

DOI:  https://doi.org/10.1002/1873-3468.14338
Nat Commun. 2022 Mar 15. 13(1): 1365

Intrinsically disordered intracellular domains control key features of the mechanically-gated ion channel PIEZO2.

Clement Verkest, Irina Schaefer, Timo A Nees, Na Wang, Juri M Jegelka, Francisco J Taberner, Stefan G Lechner.

A central question in mechanobiology is how mechanical forces acting in or on cells are transmitted to mechanically-gated PIEZO channels that convert these forces into biochemical signals. Here we examined the role of the intracellular domains of PIEZO2, which account for 25% of the channel, and demonstrate that these domains fine-tune properties such as poking and stretch-sensitivity, velocity coding and single channel conductance. Moreover, we show that the intrinsically disordered linker between the transmembrane helices twelve and thirteen (IDR5) is required for the activation of PIEZO2 by cytoskeleton-transmitted forces. The deletion of IDR5 abolishes PIEZO2-mediated inhibition of neurite outgrowth, while it only partially affected its sensitivity to cell indentation and does not alter its stretch sensitivity. Thus, we propose that PIEZO2 is a polymodal mechanosensor that detects different types of mechanical stimuli via different force transmission pathways, which highlights the importance of utilizing multiple complementary assays when investigating PIEZO function.

DOI: https://doi.org/10.1038/s41467-022-28974-6
Methods Mol Biol. 2022 ;2405 151-167

Peptide Dynamics and Metadynamics: Leveraging Enhanced Sampling Molecular Dynamics to Robustly Model Long-Timescale Transitions.

Joseph Clayton, Lokesh Baweja, Jeff Wereszczynski.

  Molecular dynamics simulations can in theory reveal the thermodynamics and kinetics of peptide conformational transitions at atomic-level resolution. However, even with modern computing power, they are limited in the timescales they can sample, which is especially problematic for peptides that are fully or partially disordered. Here, we discuss how the enhanced sampling methods accelerated molecular dynamics (aMD) and metadynamics can be leveraged in a complementary fashion to quickly explore conformational space and then robustly quantify the underlying free energy landscape. We apply these methods to two peptides that have an intrinsically disordered nature, the histone H3 and H4 N-terminal tails, and use metadynamics to compute the free energy landscape along collective variables discerned from aMD simulations. Results show that these peptides are largely disordered, with a slight preference for α-helical structures.

Keywords:  Accelerated molecular dynamics; Collective variables; Metadynamics; Peptide dynamics

DOI:  https://doi.org/10.1007/978-1-0716-1855-4_8
Methods Mol Biol. 2022 ;2405 95-113

Dynamics of Amyloid Formation from Simplified Representation to Atomistic Simulations.

Phuong Hoang Nguyen, Pierre Tufféry, Philippe Derreumaux.

  Amyloid fibril formation is an intrinsic property of short peptides, non-disease proteins, and proteins associated with neurodegenerative diseases. Aggregates of the Aβ and tau proteins, the α-synuclein protein, and the prion protein are observed in the brain of Alzheimer's, Parkinson's, and prion disease patients, respectively. Due to the transient short-range and long-range interactions of all species and their high aggregation propensities, the conformational ensemble of these devastating proteins, the exception being for the monomeric prion protein, remains elusive by standard structural biology methods in bulk solution and in lipid membranes. To overcome these limitations, an increasing number of simulations using different sampling methods and protein models have been performed. In this chapter, we first review our main contributions to the field of amyloid protein simulations aimed at understanding the early aggregation steps of short linear amyloid peptides, the conformational ensemble of the Aβ40/42 dimers in bulk solution, and the stability of Aβ aggregates in lipid membrane models. Then we focus on our studies on the interactions of amyloid peptides/inhibitors to prevent aggregation, and long amyloid sequences, including new results on a monomeric tau construct.

Keywords:  Aggregation; Amyloid; Aβ; Bulk solution; Inhibitors; Intrinsically disordered proteins; Membranes; Simulations; Tau; α-synuclein

DOI:  https://doi.org/10.1007/978-1-0716-1855-4_5
Structure. 2022 Mar 10. pii: S0969-2126(22)00049-1. [Epub ahead of print]

A "spindle and thread" mechanism unblocks p53 translation by modulating N-terminal disorder.

Margit Kaldmäe, Thibault Vosselman, Xueying Zhong, Dilraj Lama, Gefei Chen, Mihkel Saluri, Nina Kronqvist, Jia Wei Siau, Aik Seng Ng, Farid J Ghadessy, Pierre Sabatier, Borivoj Vojtesek, Médoune Sarr, Cagla Sahin, Nicklas Österlund, Leopold L Ilag, Venla A Väänänen, Saikiran Sedimbi, Marie Arsenian-Henriksson, Roman A Zubarev, Lennart Nilsson, Philip J B Koeck, Anna Rising, Axel Abelein, Nicolas Fritz, Jan Johansson, David P Lane, Michael Landreh.

  Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility." Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular "spindle and thread" mechanism unblocks protein translation in vitro.

Keywords:  intrinsically disordered proteins; protein folding; protein translation; tumor suppressor

DOI:  https://doi.org/10.1016/j.str.2022.02.013
Sci Rep. 2022 Mar 15. 12(1): 4390

Kinetic interplay between droplet maturation and coalescence modulates shape of aged protein condensates.

Adiran Garaizar, Jorge R Espinosa, Jerelle A Joseph, Rosana Collepardo-Guevara.

Biomolecular condensates formed by the process of liquid-liquid phase separation (LLPS) play diverse roles inside cells, from spatiotemporal compartmentalisation to speeding up chemical reactions. Upon maturation, the liquid-like properties of condensates, which underpin their functions, are gradually lost, eventually giving rise to solid-like states with potential pathological implications. Enhancement of inter-protein interactions is one of the main mechanisms suggested to trigger the formation of solid-like condensates. To gain a molecular-level understanding of how the accumulation of stronger interactions among proteins inside condensates affect the kinetic and thermodynamic properties of biomolecular condensates, and their shapes over time, we develop a tailored coarse-grained model of proteins that transition from establishing weak to stronger inter-protein interactions inside condensates. Our simulations reveal that the fast accumulation of strongly binding proteins during the nucleation and growth stages of condensate formation results in aspherical solid-like condensates. In contrast, when strong inter-protein interactions appear only after the equilibrium condensate has been formed, or when they accumulate slowly over time with respect to the time needed for droplets to fuse and grow, spherical solid-like droplets emerge. By conducting atomistic potential-of-mean-force simulations of NUP-98 peptides-prone to forming inter-protein [Formula: see text]-sheets-we observe that formation of inter-peptide [Formula: see text]-sheets increases the strength of the interactions consistently with the loss of liquid-like condensate properties we observe at the coarse-grained level. Overall, our work aids in elucidating fundamental molecular, kinetic, and thermodynamic mechanisms linking the rate of change in protein interaction strength to condensate shape and maturation during ageing.

DOI: https://doi.org/10.1038/s41598-022-08130-2