bims-indpro Biomed News
on Intrinsically disordered proteins
Issue of 2022‒06‒12
twenty-one papers selected by
Sara Mingu
Johannes Gutenberg University
  1. A modular approach to map out the conformational landscapes of unbound intrinsically disordered proteins.
  2. PQBP1: The Key to Intellectual Disability, Neurodegenerative Diseases, and Innate Immunity.
  3. BIAPSS: A Comprehensive Physicochemical Analyzer of Proteins Undergoing Liquid-Liquid Phase Separation.
  4. Conformational dynamics in the disordered region of human CPEB3 linked to memory consolidation.
  5. Negatively charged disordered regions are prevalent and functionally important across proteomes.
  6. JMJD3 intrinsically disordered region links the 3D-genome structure to TGFβ-dependent transcription activation.
  7. Phase separation of Ddx3xb helicase regulates maternal-to-zygotic transition in zebrafish.
  8. Alpha-Synuclein Aggregation Pathway in Parkinson's Disease: Current Status and Novel Therapeutic Approaches.
  9. Camouflaged Fluorescent Silica Nanoparticles Target Aggregates and Condensates of the Amyloidogenic Protein Tau.
  10. Extent of intrinsic disorder and NMR chemical shift assignments of the distal N-termini from human TRPV1, TRPV2 and TRPV3 ion channels.
  11. Controlling synthetic membraneless organelles by a red-light-dependent singlet oxygen-generating protein.
  12. α-Synuclein in the Synaptic Vesicle Liquid Phase: Active Player or Passive Bystander?
  13. Exploring the Conformations and Binding Location of HMGA2·DNA Complexes Using Ion Mobility Spectrometry and 193 nm Ultraviolet Photodissociation Mass Spectrometry.
  14. Plant Protein Disorder: Spatial Regulation, Broad Specificity, Switch of Signaling and Physiological Status.
  15. 40S hnRNP particles are a novel class of nuclear biomolecular condensates.
  16. The Hydrophilic Loop of Arabidopsis PIN1 Auxin Efflux Carrier Harbors Hallmarks of an Intrinsically Disordered Protein.
  17. Not Only Systemin: Prosystemin Harbors Other Active Regions Able to Protect Tomato Plants.
  18. Phosphatidylserine and Phosphatidylethanolamine Asymmetry Have a Negligible Effect on the Global Structure, Dynamics, and Interactions of the KRAS Lipid Anchor.
  19. NMR resonance assignments of the DNA binding domain of mouse Junctophilin-2.
  20. The Disordered EZH2 Loop: Atomic Level Characterization by 1HN- and 1Hα-Detected NMR Approaches, Interaction with the Long Noncoding HOTAIR RNA.
  21. Copper(II) Binding to the Intrinsically Disordered C-Terminal Peptide of SARS-CoV-2 Virulence Factor Nsp1.
  1. Proc Natl Acad Sci U S A. 2022 Jun 07. 119(23): e2113572119
    A modular approach to map out the conformational landscapes of unbound intrinsically disordered proteins.
    Thinh D N Luong, Suhani Nagpal, Mourad Sadqi, Victor Muñoz.
      SignificanceIntrinsically disordered proteins have the unique ability to morph in response to multiple partners and thereby process sophisticated inputs and outputs. It is, however, a mystery whether their response is passive, that is, entirely determined by the partner, or controlled via an internal, yet unknown, folding mechanism. Here we introduce an approach to examine this key question and demonstrate its potential by dissecting the conformational properties of the partially disordered protein NCBD and obtaining important clues about how it performs its biological function.
    Keywords:  conformational rheostat; folding landscapes; folding upon binding; intrinsically disordered proteins
    DOI:  https://doi.org/10.1073/pnas.2113572119
  2. Int J Mol Sci. 2022 Jun 02. pii: 6227. [Epub ahead of print]23(11):
    PQBP1: The Key to Intellectual Disability, Neurodegenerative Diseases, and Innate Immunity.
    Hikari Tanaka, Hitoshi Okazawa.
      The idea that a common pathology underlies various neurodegenerative diseases and dementias has attracted considerable attention in the basic and medical sciences. Polyglutamine binding protein-1 (PQBP1) was identified in 1998 after a molecule was predicted to bind to polyglutamine tract amino acid sequences, which are associated with a family of neurodegenerative disorders called polyglutamine diseases. Hereditary gene mutations of PQBP1 cause intellectual disability, whereas acquired loss of function of PQBP1 contributes to dementia pathology. PQBP1 functions in innate immune cells as an intracellular receptor that recognizes pathogens and neurodegenerative proteins. It is an intrinsically disordered protein that generates intracellular foci, similar to other neurodegenerative disease proteins such as TDP43, FUS, and hnRNPs. The knowledge accumulated over more than 20 years has given rise to a new concept that shifts in the equilibrium between physiological and pathological processes have their basis in the dysregulation of common protein structure-linked molecular mechanisms.
    Keywords:  PQBP1; innate immunity; intellectual disability; intrinsically disordered protein; neurodegenerative diseases
    DOI:  https://doi.org/10.3390/ijms23116227
  3. Int J Mol Sci. 2022 May 31. pii: 6204. [Epub ahead of print]23(11):
    BIAPSS: A Comprehensive Physicochemical Analyzer of Proteins Undergoing Liquid-Liquid Phase Separation.
    Aleksandra E Badaczewska-Dawid, Vladimir N Uversky, Davit A Potoyan.
      The liquid-liquid phase separation (LLPS) of biomolecules is a phenomenon which is nowadays recognized as the driving force for the biogenesis of numerous functional membraneless organelles and cellular bodies. The interplay between the protein primary sequence and phase separation remains poorly understood, despite intensive research. To uncover the sequence-encoded signals of protein capable of undergoing LLPS, we developed a novel web platform named BIAPSS (Bioinformatics Analysis of LLPS Sequences). This web server provides on-the-fly analysis, visualization, and interpretation of the physicochemical and structural features for the superset of curated LLPS proteins.
    Keywords:  intrinsically disordered proteins; liquid–liquid phase separation; membraneless organelles; proteins with low complexity
    DOI:  https://doi.org/10.3390/ijms23116204
  4. BMC Biol. 2022 06 03. 20(1): 129
    Conformational dynamics in the disordered region of human CPEB3 linked to memory consolidation.
    D Ramírez de Mingo, D Pantoja-Uceda, R Hervás, M Carrión-Vázquez, D V Laurents.
      BACKGROUND: Current understanding of the molecular basis of memory consolidation points to an important function of amyloid formation by neuronal-specific isoforms of the cytoplasmic polyadenylation element binding (CPEB) protein family. In particular, CPEB is thought to promote memory persistence through formation of self-sustaining prion-like amyloid assemblies at synapses, mediated by its intrinsically disordered region (IDR) and leading to permanent physical alterations at the basis of memory persistence. Although the molecular mechanisms by which amyloid formation takes place in CPEB have been described in invertebrates, the way amyloid formation occurs in the human homolog CPEB3 (hCPEB3) remains unclear. Here, we characterize by NMR spectroscopy the atomic level conformation and ps-ms dynamics of the 426-residue IDR of hCPEB3, which has been associated with episodic memory in humans.RESULTS: We show that the 426-residue N-terminal region of hCPEB3 is a dynamic, intrinsically disordered region (IDR) which lacks stable folded structures. The first 29 residues, M1QDDLLMDKSKTQPQPQQQQRQQQQPQP29, adopt a helical + disordered motif, and residues 86-93: P83QQPPPP93, and 166-175: P166PPPAPAPQP175 form polyproline II (PPII) helices. The (VG)5 repeat motif is completely disordered, and residues 200-250 adopt three partially populated α-helices. Residues 345-355, which comprise the nuclear localization signal (NLS), form a modestly populated α-helix which may mediate STAT5B binding. These findings allow us to suggest a model for nascent hCPEB3 structural transitions at single residue resolution, advancing that amyloid breaker residues, like proline, are a key difference between functional versus pathological amyloids.
    CONCLUSION: Our NMR spectroscopic analysis of hCPEB3 provides insights into the first structural transitions involved in protein-protein and protein-mRNA interactions. The atomic level understanding of these structural transitions involved in hCPEB3 aggregation is a key first step toward understanding memory persistence in humans, as well as sequence features that differentiate beneficial amyloids from pathological ones.
    AREAS: Biophysics, Structural Biology, Biochemistry & Neurosciences.
    Keywords:  Intrinsically disordered proteins; Memory consolidation; NMR spectroscopy
    DOI:  https://doi.org/10.1186/s12915-022-01310-6
  5. J Mol Biol. 2022 May 31. pii: S0022-2836(22)00252-2. [Epub ahead of print] 167660
    Negatively charged disordered regions are prevalent and functionally important across proteomes.
    Lavi S Bigman, Junji Iwahara, Yaakov Levy.
      Intrinsically disordered regions (IDRs) of proteins are often characterized by a high fraction of charged residues, but differ in their overall net charge and in the organization of the charged residues. The function-encoding information stored via IDR charge composition and organization remains elusive. Here, we aim to decipher the sequence-function relationship in IDRs by presenting a comprehensive bioinformatic analysis of the charge properties of IDRs in the human, mouse, and yeast proteomes. About 50% of the proteins comprise at least a single IDR, which is either positively or negatively charged. Highly negatively charged IDRs are longer and possess greater net charge per residue compared with highly positively charged IDRs. A striking difference between positively and negatively charged IDRs is the characteristics of the repeated units, specifically, of consecutive Lys or Arg residues (K/R repeats) and Asp or Glu (D/E repeats) residues. D/E repeats are found to be about five times longer than K/R repeats, with the longest found containing 49 residues. Long stretches of consecutive D and E are found to be more prevalent in nucleic acid-related proteins. They are less common in prokaryotes, and in eukaryotes their abundance increases with genome size. The functional role of D/E repeats and the profound differences between them and K/R repeats are discussed.
    Keywords:  Disordered regions; electrostatics; polyampholytes; repeat sequences, D/E repeat
    DOI:  https://doi.org/10.1016/j.jmb.2022.167660
  6. Nat Commun. 2022 Jun 07. 13(1): 3263
    JMJD3 intrinsically disordered region links the 3D-genome structure to TGFβ-dependent transcription activation.
    Marta Vicioso-Mantis, Raquel Fueyo, Claudia Navarro, Sara Cruz-Molina, Wilfred F J van Ijcken, Elena Rebollo, Álvaro Rada-Iglesias, Marian A Martínez-Balbás.
      Enhancers are key regulatory elements that govern gene expression programs in response to developmental signals. However, how multiple enhancers arrange in the 3D-space to control the activation of a specific promoter remains unclear. To address this question, we exploited our previously characterized TGFβ-response model, the neural stem cells, focusing on a ~374 kb locus where enhancers abound. Our 4C-seq experiments reveal that the TGFβ pathway drives the assembly of an enhancer-cluster and precise gene activation. We discover that the TGFβ pathway coactivator JMJD3 is essential to maintain these structures. Using live-cell imaging techniques, we demonstrate that an intrinsically disordered region contained in JMJD3 is involved in the formation of phase-separated biomolecular condensates, which are found in the enhancer-cluster. Overall, in this work we uncover novel functions for the coactivator JMJD3, and we shed light on the relationships between the 3D-conformation of the chromatin and the TGFβ-driven response during mammalian neurogenesis.
    DOI:  https://doi.org/10.1038/s41467-022-30614-y
  7. Cell Res. 2022 Jun 03.
    Phase separation of Ddx3xb helicase regulates maternal-to-zygotic transition in zebrafish.
    Boyang Shi, Jian Heng, Jia-Yi Zhou, Ying Yang, Wan-Ying Zhang, Magdalena J Koziol, Yong-Liang Zhao, Pilong Li, Feng Liu, Yun-Gui Yang.
      Vertebrate embryogenesis involves a conserved and fundamental process, called the maternal-to-zygotic transition (MZT), which marks the switch from a maternal factors-dominated state to a zygotic factors-driven state. Yet the precise mechanism underlying MZT remains largely unknown. Here we report that the RNA helicase Ddx3xb in zebrafish undergoes liquid-liquid phase separation (LLPS) via its N-terminal intrinsically disordered region (IDR), and an increase in ATP content promotes the condensation of Ddx3xb during MZT. Mutant form of Ddx3xb losing LLPS ability fails to rescue the developmental defect of Ddx3xb-deficient embryos. Interestingly, the IDR of either FUS or hnRNPA1 can functionally replace the N-terminal IDR in Ddx3xb. Phase separation of Ddx3xb facilitates the unwinding of 5' UTR structures of maternal mRNAs to enhance their translation. Our study reveals an unprecedent mechanism whereby the Ddx3xb phase separation regulates MZT by promoting maternal mRNA translation.
    DOI:  https://doi.org/10.1038/s41422-022-00655-5
  8. Cells. 2022 May 24. pii: 1732. [Epub ahead of print]11(11):
    Alpha-Synuclein Aggregation Pathway in Parkinson's Disease: Current Status and Novel Therapeutic Approaches.
    Marija Vidović, Milena G Rikalovic.
      Following Alzheimer's, Parkinson's disease (PD) is the second-most common neurodegenerative disorder, sharing an unclear pathophysiology, a multifactorial profile, and massive social costs worldwide. Despite this, no disease-modifying therapy is available. PD is tightly associated with α-synuclein (α-Syn) deposits, which become organised into insoluble, amyloid fibrils. As a typical intrinsically disordered protein, α-Syn adopts a monomeric, random coil conformation in an aqueous solution, while its interaction with lipid membranes drives the transition of the molecule part into an α-helical structure. The central unstructured region of α-Syn is involved in fibril formation by converting to well-defined, β-sheet rich secondary structures. Presently, most therapeutic strategies against PD are focused on designing small molecules, peptides, and peptidomimetics that can directly target α-Syn and its aggregation pathway. Other approaches include gene silencing, cell transplantation, stimulation of intracellular clearance with autophagy promoters, and degradation pathways based on immunotherapy of amyloid fibrils. In the present review, we sum marise the current advances related to α-Syn aggregation/neurotoxicity. These findings present a valuable arsenal for the further development of efficient, nontoxic, and non-invasive therapeutic protocols for disease-modifying therapy that tackles disease onset and progression in the future.
    Keywords:  disaggregators; high throughput anti-aggregation drug screening; intrinsically disordered proteins; protein misfolding; rationally designed peptidomimetics; structure/function relationship; synucleinopathies; α-synuclein oligomers and fibrils
    DOI:  https://doi.org/10.3390/cells11111732
  9. Bioconjug Chem. 2022 Jun 10.
    Camouflaged Fluorescent Silica Nanoparticles Target Aggregates and Condensates of the Amyloidogenic Protein Tau.
    Carlo Giorgio Barracchia, Francesca Parolini, Angela Volpe, Daniele Gori, Francesca Munari, Stefano Capaldi, Mariapina D'Onofrio, Michael Assfalg.
      Intrinsically disordered proteins (IDPs) are increasingly found to be associated with irreversible neurodegenerative disorders. The protein tau is a prototypical IDP whose abnormal aggregation into insoluble filaments is a major hallmark of Alzheimer's disease. The view has emerged that aggregation may proceed via alternative pathways involving oligomeric intermediates or phase-separated liquid droplets. Nanoparticles (NPs) offer significant potential for probing the mechanisms of protein fibrillation and may be capable of redirecting conformational transitions. Here, we camouflaged dye-doped silica NPs through functionalization with tau molecules to impart them the ability to associate with protein assemblies such as aggregates or condensates. The prepared NP-tau conjugates showed little influence on the aggregation kinetics and morphology of filamentous aggregates of tau but were found to associate with the filaments. Moreover, NP-tau conjugates were recruited and concentrated into polyanion-induced condensates of tau, driven by multivalent electrostatic interactions, thereby illuminating liquid droplets and their time-dependent transformation, as observed by fluorescence microscopy. NP-tau conjugates were capable of entering human neuroglioma cells and were not cytotoxic. Hence, we propose that NP-tau conjugates could serve as nanotracers for in vitro and in-cell studies to target and visualize tau assemblies and condensates, contributing to an explanation for the molecular mechanisms of abnormal protein aggregation.
    DOI:  https://doi.org/10.1021/acs.bioconjchem.2c00168
  10. Biomol NMR Assign. 2022 Jun 06.
    Extent of intrinsic disorder and NMR chemical shift assignments of the distal N-termini from human TRPV1, TRPV2 and TRPV3 ion channels.
    Christoph Wiedemann, Benedikt Goretzki, Zoe N Merz, Frederike Tebbe, Pauline Schmitt, Ute A Hellmich.
      The mammalian Transient Receptor Potential Vanilloid (TRPV) channels are a family of six tetrameric ion channels localized at the plasma membrane. The group I members of the family, TRPV1 through TRPV4, are heat-activated and exhibit remarkable polymodality. The distal N-termini of group I TRPV channels contain large intrinsically disordered regions (IDRs), ranging from ~ 75 amino acids (TRPV2) to ~ 150 amino acids (TRPV4), the vast majority of which is invisible in the structural models published so far. These IDRs provide important binding sites for cytosolic partners, and their deletion is detrimental to channel activity and regulation. Recently, we reported the NMR backbone assignments of the distal TRPV4 N-terminus and noticed some discrepancies between the extent of disorder predicted solely based on protein sequence and from experimentally determined chemical shifts. Thus, for an analysis of the extent of disorder in the distal N-termini of all group I TRPV channels, we now report the NMR assignments for the human TRPV1, TRPV2 and TRPV3 IDRs.
    Keywords:  Intrinsically disordered protein; Ion channel; Regulatory domain; Structural dynamics; TRP vanilloid; Transient receptor potential
    DOI:  https://doi.org/10.1007/s12104-022-10093-4
  11. Nat Commun. 2022 Jun 09. 13(1): 3197
    Controlling synthetic membraneless organelles by a red-light-dependent singlet oxygen-generating protein.
    Manjia Li, Byung Min Park, Xin Dai, Yingjie Xu, Jinqing Huang, Fei Sun.
      Membraneless organelles (MLOs) formed via protein phase separation have great implications for both physiological and pathological processes. However, the inability to precisely control the bioactivities of MLOs has hindered our understanding of their roles in biology, not to mention their translational applications. Here, by combining intrinsically disordered domains such as RGG and mussel-foot proteins, we create an in cellulo protein phase separation system, of which various biological activities can be introduced via metal-mediated protein immobilization and further controlled by the water-soluble chlorophyll protein (WSCP)-a remarkably stable, red-light-responsive singlet oxygen generator. The WSCP-laden protein condensates undergo a liquid-to-solid phase transition on light exposure, due to oxidative crosslinking, providing a means to control catalysis within synthetic MLOs. Moreover, these photoresponsive condensates, which retain the light-induced phase-transition behavior in living cells, exhibit marked membrane localization, reminiscent of the semi-membrane-bound compartments like postsynaptic densities in nervous systems. Together, this engineered system provides an approach toward controllable synthetic MLOs and, alongside its light-induced phase transition, may well serve to emulate and explore the aging process at the subcellular or even molecular level.
    DOI:  https://doi.org/10.1038/s41467-022-30933-0
  12. Front Mol Biosci. 2022 ;9 891508
    α-Synuclein in the Synaptic Vesicle Liquid Phase: Active Player or Passive Bystander?
    Lennart Brodin, Dragomir Milovanovic, Silvio O Rizzoli, Oleg Shupliakov.
      The protein α-synuclein, which is well-known for its links to Parkinson's Disease, is associated with synaptic vesicles (SVs) in nerve terminals. Despite intensive studies, its precise physiological function remains elusive. Accumulating evidence indicates that liquid-liquid phase separation takes part in the assembly and/or maintenance of different synaptic compartments. The current review discusses recent data suggesting α-synuclein as a component of the SV liquid phase. We also consider possible implications of these data for disease.
    Keywords:  liquid-liquid phase separation (LLPS); synapse; synapsin; synaptic vesicle; synaptic vesicle clustering; α-Synuclein
    DOI:  https://doi.org/10.3389/fmolb.2022.891508
  13. J Am Soc Mass Spectrom. 2022 Jun 10.
    Exploring the Conformations and Binding Location of HMGA2·DNA Complexes Using Ion Mobility Spectrometry and 193 nm Ultraviolet Photodissociation Mass Spectrometry.
    Sarah N Sipe, Kevin Jeanne Dit Fouque, Alyssa Garabedian, Fenfei Leng, Francisco Fernandez-Lima, Jennifer S Brodbelt.
      Although it is widely accepted that protein function is largely dependent on its structure, intrinsically disordered proteins (IDPs) lack defined structure but are essential in proper cellular processes. Mammalian high mobility group proteins (HMGA) are one such example of IDPs that perform a number of crucial nuclear activities and have been highly studied due to their involvement in the proliferation of a variety of disease and cancers. Traditional structural characterization methods have had limited success in understanding HMGA proteins and their ability to coordinate to DNA. Ion mobility spectrometry and mass spectrometry provide insights into the diversity and heterogeneity of structures adopted by IDPs and are employed here to interrogate HMGA2 in its unbound states and bound to two DNA hairpins. The broad distribution of collision cross sections observed for the apo-protein are restricted when HMGA2 is bound to DNA, suggesting that increased protein organization is promoted in the holo-form. Ultraviolet photodissociation was utilized to probe the changes in structures for the compact and elongated structures of HMGA2 by analyzing backbone cleavage propensities and solvent accessibility based on charge-site analysis, which revealed a spectrum of conformational possibilities. Namely, preferential binding of the DNA hairpins with the second of three AT-hooks of HMGA2 is suggested based on the suppression of backbone fragmentation and distribution of DNA-containing protein fragments.
    DOI:  https://doi.org/10.1021/jasms.2c00083
  14. Front Plant Sci. 2022 ;13 904446
    Plant Protein Disorder: Spatial Regulation, Broad Specificity, Switch of Signaling and Physiological Status.
    An-Shan Hsiao.
      
    Keywords:  conformation; intrinsically disordered proteins (IDPs); intrinsically disordered region (IDR); liquid–liquid phase separation; protein disorder; spatial regulation of signaling
    DOI:  https://doi.org/10.3389/fpls.2022.904446
  15. Nucleic Acids Res. 2022 Jun 10. pii: gkac457. [Epub ahead of print]
    40S hnRNP particles are a novel class of nuclear biomolecular condensates.
    Michal Domanski, Emil Dedic, Maria Escura Pérez, Antoine Cléry, Sébastien Campagne, Anne-Christine Uldry, Sophie Braga, Manfred Heller, Julius Rabl, Pavel Afanasyev, Daniel Boehringer, Jiří Nováček, Frédéric T Allain, Oliver Mühlemann.
      Heterogenous nuclear ribonucleoproteins (hnRNPs) are abundant proteins implicated in various steps of RNA processing that assemble on nuclear RNA into larger complexes termed 40S hnRNP particles. Despite their initial discovery 55 years ago, our understanding of these intriguing macromolecular assemblies remains limited. Here, we report the biochemical purification of native 40S hnRNP particles and the determination of their complete protein composition by label-free quantitative mass spectrometry, identifying A-group and C-group hnRNPs as the major protein constituents. Isolated 40S hnRNP particles dissociate upon RNA digestion and can be reconstituted in vitro on defined RNAs in the presence of the individual protein components, demonstrating a scaffolding role for RNA in nucleating particle formation. Finally, we revealed their nanometer scale, condensate-like nature, promoted by intrinsically disordered regions of A-group hnRNPs. Collectively, we identify nuclear 40S hnRNP particles as novel dynamic biomolecular condensates.
    DOI:  https://doi.org/10.1093/nar/gkac457
  16. Int J Mol Sci. 2022 Jun 06. pii: 6352. [Epub ahead of print]23(11):
    The Hydrophilic Loop of Arabidopsis PIN1 Auxin Efflux Carrier Harbors Hallmarks of an Intrinsically Disordered Protein.
    Veronika Bilanovičová, Nikola Rýdza, Lilla Koczka, Martin Hess, Elena Feraru, Jiří Friml, Tomasz Nodzyński.
      Much of plant development depends on cell-to-cell redistribution of the plant hormone auxin, which is facilitated by the plasma membrane (PM) localized PIN FORMED (PIN) proteins. Auxin export activity, developmental roles, subcellular trafficking, and polarity of PINs have been well studied, but their structure remains elusive besides a rough outline that they contain two groups of 5 alpha-helices connected by a large hydrophilic loop (HL). Here, we focus on the PIN1 HL as we could produce it in sufficient quantities for biochemical investigations to provide insights into its secondary structure. Circular dichroism (CD) studies revealed its nature as an intrinsically disordered protein (IDP), manifested by the increase of structure content upon thermal melting. Consistent with IDPs serving as interaction platforms, PIN1 loops homodimerize. PIN1 HL cytoplasmic overexpression in Arabidopsis disrupts early endocytic trafficking of PIN1 and PIN2 and causes defects in the cotyledon vasculature formation. In summary, we demonstrate that PIN1 HL has an intrinsically disordered nature, which must be considered to gain further structural insights. Some secondary structures may form transiently during pairing with known and yet-to-be-discovered interactors.
    Keywords:  PIN1; dimerization; hydrophilic hoop; intrinsic disorder; subcellular trafficking
    DOI:  https://doi.org/10.3390/ijms23116352
  17. Front Plant Sci. 2022 ;13 887674
    Not Only Systemin: Prosystemin Harbors Other Active Regions Able to Protect Tomato Plants.
    Donata Molisso, Mariangela Coppola, Martina Buonanno, Ilaria Di Lelio, Anna Maria Aprile, Emma Langella, Maria Manuela Rigano, Silvana Francesca, Pasquale Chiaiese, Gianna Palmieri, Rosarita Tatè, Martina Sinno, Eleonora Barra, Andrea Becchimanzi, Simona Maria Monti, Francesco Pennacchio, Rosa Rao.
      Prosystemin is a 200-amino acid precursor expressed in Solanaceae plants which releases at the C-terminal part a peptidic hormone called Systemin in response to wounding and herbivore attack. We recently showed that Prosystemin is not only a mere scaffold of Systemin but, even when deprived of Systemin, is biologically active. These results, combined with recent discoveries that Prosystemin is an intrinsically disordered protein containing disordered regions within its sequence, prompted us to investigate the N-terminal portions of the precursor, which contribute to the greatest disorder within the sequence. To this aim, PS1-70 and PS1-120 were designed, produced, and structurally and functionally characterized. Both the fragments, which maintained their intrinsic disorder, were able to induce defense-related genes and to protect tomato plants against Botrytis cinerea and Spodoptera littoralis larvae. Intriguingly, the biological activity of each of the two N-terminal fragments and of Systemin is similar but not quite the same and does not show any toxicity on experimental non-targets considered. These regions account for different anti-stress activities conferred to tomato plants by their overexpression. The two N-terminal fragments identified in this study may represent new promising tools for sustainable crop protection.
    Keywords:  bioactivity; endogenous defense; fragments; insect herbivores; natively unfolded; not direct toxicity effect; phytopathogenic fungi; tomato protection
    DOI:  https://doi.org/10.3389/fpls.2022.887674
  18. J Phys Chem B. 2022 Jun 10.
    Phosphatidylserine and Phosphatidylethanolamine Asymmetry Have a Negligible Effect on the Global Structure, Dynamics, and Interactions of the KRAS Lipid Anchor.
    Mussie K Araya, Alemayehu A Gorfe.
      The intrinsically disordered C-terminus of the prominent oncogenic protein KRAS-4B (KRAS) selectively interacts and clusters with phosphatidylserine (PS) lipids in the plasma membrane (PM). This 11-residue segment, called tK, contains a polybasic domain (PBD) of six contiguous lysine residues and a farnesylated cysteine. Previous molecular dynamics (MD) simulation studies of tK in phosphatidylcholine (PC)/PS bilayers have suggested that backbone conformational dynamics modulate tK-PS interactions. These simulations have been conducted in symmetric membranes whereas the PM is compositionally asymmetric, with the inner leaflet, where KRAS is localized, being enriched with PS and phosphatidylethanolamine (PE) lipids. To examine if bilayer asymmetry affects tK conformational dynamics and interaction with lipids, we conducted two 10 μs long MD simulations of tK bound to a PC/PS and a PC/PS/PE bilayer in which the PS and PE lipids are distributed in one leaflet. We found that, first, these compositional asymmetries caused differences in acyl chain dynamics between leaflets, but the equilibrium structural and dynamic properties of the two asymmetric bilayers are similar; second, in both systems tK is highly dynamic and samples at least two distinct conformational states; third, PS-tK hydrogen-bonding interactions vary with peptide backbone conformations, and lysine side chains in the PBD predominantly interact with the serine oxygens of PS. These results are in good agreement with previous observations of tK in symmetric membranes. The effects of POPS asymmetry or the presence of POPE on tK are limited to modulating the relative contribution of individual side chains to interactions with lipids and redistributing conformational substates. Additional observations include the larger flexibility of tK in the current simulations, which we attribute to the longer duration of the simulations and the use of the CHARMM36m force field, which more accurately models intrinsically disordered peptides such as tK.
    DOI:  https://doi.org/10.1021/acs.jpcb.2c01253
  19. Biomol NMR Assign. 2022 Jun 04.
    NMR resonance assignments of the DNA binding domain of mouse Junctophilin-2.
    Liping Yu, Duane D Hall, Weiyang Zhao, Long-Sheng Song.
      Junctophilin-2 (JP2) is a critical structural protein in the heart by stabilizing junctional membrane complexes between the plasma membrane and sarcoplasmic reticula responsible for precise Ca2+ regulation. Such complexes are essential for efficient cardiomyocyte contraction and adaptation to altered cardiac workload conditions. Mutations in the JPH2 gene that encodes JP2 are associated with inherited cardiomyopathies and arrhythmias, and disruption of JP2 function is lethal. Interestingly, cardiac stress promotes the proteolytic cleavage of JP2 that triggers the translocation of its N-terminal fragment into the nucleus to repress maladaptive gene transcription. We previously found that the central region of JP2 is responsible for mediating direct DNA binding interactions. Recent structural studies indicate that this region serves as a structural role in the cytosolic form of JP2 by folding into a single continuous α-helix. However, the structural basis of how this DNA-binding domain interacts with DNA is not known. Here, we report the backbone and sidechain assignments of the DNA-binding domain (residues 331-413) of mouse JP2. These assignments reveal that the JP2 DNA binding domain is an intrinsically disordered protein and contains two α-helices located in the C-terminal portion of the protein. Moreover, this protein binds to DNA in a similar manner to that shown previously by electrophoretic mobility shift assays. Therefore, these assignments provide a framework for further structural studies into the interaction of this JP2 domain with DNA for the elucidation of transcriptional regulation of stress-responsive genes as well as its role in the stabilization of junctional membrane complexes.
    Keywords:  Cardiomyocyte; Cardiomyopathies; DNA binding; Excitation–contraction coupling; Junctophilin; Junctophilin-2
    DOI:  https://doi.org/10.1007/s12104-022-10091-6
  20. Int J Mol Sci. 2022 May 30. pii: 6150. [Epub ahead of print]23(11):
    The Disordered EZH2 Loop: Atomic Level Characterization by 1HN- and 1Hα-Detected NMR Approaches, Interaction with the Long Noncoding HOTAIR RNA.
    Csenge Lilla Szabó, Beáta Szabó, Fanni Sebák, Wolfgang Bermel, Agnes Tantos, Andrea Bodor.
      The 96-residue-long loop of EZH2 is proposed to play a role in the interaction with long non-coding RNAs (lncRNAs) and to contribute to EZH2 recruitment to the chromatin. However, molecular details of RNA recognition have not been described so far. Cellular studies have suggested that phosphorylation of the Thr345 residue localized in this loop influences RNA binding; however, no mechanistic explanation has been offered. To address these issues, a systematic NMR study was performed. As the 1HN-detected NMR approach presents many challenges under physiological conditions, our earlier developed, as well as improved, 1Hα-detected experiments were used. As a result of the successful resonance assignment, the obtained chemical shift values indicate the highly disordered nature of the EZH2 loop, with some nascent helical tendency in the Ser407-Ser412 region. Further investigations conducted on the phosphomimetic mutant EZH2T345D showed that the mutation has only a local effect, and that the loop remains disordered. On the other hand, the mutation influences the cis/trans Pro346 equilibrium. Interactions of both the wild-type and the phosphomimetic mutant with the lncRNA HOTAIR140 (1-140 nt) highlight that the Thr367-Ser375 region is affected. This segment does not resemble any of the previously reported RNA-binding motifs, therefore the identified binding region is unique. As no structural changes occur in the EZH2 loop upon RNA binding, we can consider the protein-RNA interaction as a "fuzzy" complex.
    Keywords:  1Hα detected NMR; EZH2; HOTAIR; IDP–RNA interaction; fuzzy complex; intrinsically disordered proteins
    DOI:  https://doi.org/10.3390/ijms23116150
  21. Inorg Chem. 2022 Jun 05.
    Copper(II) Binding to the Intrinsically Disordered C-Terminal Peptide of SARS-CoV-2 Virulence Factor Nsp1.
    Maryann Morales, Raheleh Ravanfar, Paul H Oyala, Harry B Gray, Jay R Winkler.
      The first encoded SARS-CoV-2 protein (Nsp1) binds to the human 40S ribosome and blocks synthesis of host proteins, thereby inhibiting critical elements of the innate immune response. The final 33 residues of the natively unstructured Nsp1 C-terminus adopt a helix-turn-helix geometry upon binding to the ribosome. We have characterized the fluctuating conformations of this peptide using circular dichroism spectroscopy along with measurements of tryptophan fluorescence and energy transfer. Tryptophan fluorescence decay kinetics reveal that copper(II) binds to the peptide at micromolar concentrations, and electron paramagnetic resonance spectroscopy indicates that the metal ion coordinates to the lone histidine residue.
    DOI:  https://doi.org/10.1021/acs.inorgchem.2c01329
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