bims-indpro 2022-08-07 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022‒08‒07
fifteen papers selected by
Sara Mingu
Johannes Gutenberg University

Self-assembly of globular proteins with intrinsically disordered protein polyelectrolytes and block copolymers.
Remodeling of the Plasma Membrane by Surface-Bound Protein Monomers and Oligomers: The Critical Role of Intrinsically Disordered Regions.
15N-Detected TROSY NMR experiments to study large disordered proteins in high-field magnets.
Integrative Conformational Ensembles of Sic1 Using Different Initial Pools and Optimization Methods.
Native mass spectrometry for the investigation of protein structural (dis)order.
Protein Condensate Formation via Controlled Multimerization of Intrinsically Disordered Sequences.
Intrinsically disordered BMP4 morphogen and the beak of the finch: Co-option of an ancient axial patterning system.
Molecular dynamics simulations of an α-synuclein NAC domain fragment with a ff14IDPSFF IDP-specific force field suggest β-sheet intermediate states of fibrillation.
Remote communication between unstructured and structured regions of Bcl-2 tunes its ligand binding capacity: Mechanistic insights.
Accurate Description of Solvent-Exposed Salt Bridges with a Non-polarizable Force Field Incorporating Solvent Effects.
Structural and biophysical properties of FopA, a major outer membrane protein of Francisella tularensis.
Review: Investigating the aggregation of amyloid beta with surface plasmon resonance: Do different approaches yield different results?
Lipid-driven condensation and interfacial ordering of FUS.
Designer D-peptides targeting the N-terminal region of α-synuclein to prevent parkinsonian-associated fibrilization and cytotoxicity.
Regulation of TIA-1 Condensates: Zn2+ and RGG Motifs Promote Nucleic Acid Driven LLPS and Inhibit Irreversible Aggregation.

Soft Matter. 2022 Aug 01.

Self-assembly of globular proteins with intrinsically disordered protein polyelectrolytes and block copolymers.

Justin M Horn, Yuncan Zhu, So Yeon Ahn, Allie C Obermeyer.

Intrinsically disordered polypeptides are a versatile class of materials, combining the biocompatibility of peptides with the disordered structure and diverse phase behaviors of synthetic polymers. Synthetic polyelectrolytes are capable of complex phase behavior when mixed with oppositely charged polyelectrolytes, facilitating nanoparticle formation and bulk phase separation. However, there has been limited exploration of intrinsically disordered protein polyelectrolytes as potential bio-based replacements for synthetic polyelectrolytes. Here, we produce negatively charged, intrinsically disordered polypeptides, capable of high-yield expression in E. coli and use this intrinsically disordered peptide to produce entirely protein-based polyelectrolyte complexes. The complexes display rich phase behavior, showing sensitivity to charge density, salt concentration, temperature, and charge fraction. We characterize this behavior through a combination of turbidity assays, dynamic light scattering, and transmission electron microscopy. The robust expression profile and stimuli-responsive phase behavior of the intrinsically disordered peptides demonstrates their potential as easily producible, biocompatible substitutes for synthetic polyelectrolytes.

DOI: https://doi.org/10.1039/d2sm00415a
J Membr Biol. 2022 Aug 05.

Remodeling of the Plasma Membrane by Surface-Bound Protein Monomers and Oligomers: The Critical Role of Intrinsically Disordered Regions.

Mussie K Araya, Yong Zhou, Alemayehu A Gorfe.

  The plasma membrane (PM) of cells is a dynamic structure whose morphology and composition is in constant flux. PM morphologic changes are particularly relevant for the assembly and disassembly of signaling platforms involving surface-bound signaling proteins, as well as for many other mechanochemical processes that occur at the PM surface. Surface-bound membrane proteins (SBMP) require efficient association with the PM for their function, which is often achieved by the coordinated interactions of intrinsically disordered regions (IDRs) and globular domains with membrane lipids. This review focuses on the role of IDR-containing SBMPs in remodeling the composition and curvature of the PM. The ability of IDR-bearing SBMPs to remodel the Gaussian and mean curvature energies of the PM is intimately linked to their ability to sort subsets of phospholipids into nanoclusters. We therefore discuss how IDRs of many SBMPs encode lipid-binding specificity or facilitate cluster formation, both of which increase their membrane remodeling capacity, and how SBMP oligomers alter membrane shape by monolayer surface area expansion and molecular crowding.

Keywords:  Intrinsically disordered regions; Lipid sorting; Membrane curvature; Membrane remodeling; Surface-bound membrane proteins

DOI:  https://doi.org/10.1007/s00232-022-00256-8
Chem Commun (Camb). 2022 Aug 03.

15N-Detected TROSY NMR experiments to study large disordered proteins in high-field magnets.

Abhinav Dubey, Thibault Viennet, Sandeep Chhabra, Koh Takeuchi, Hee-Chan Seo, Wolfgang Bermel, Dominique P Frueh, Haribabu Arthanari.

Intrinsically disordered regions (IDRs) of proteins are critical in the regulation of biological processes but difficult to study structurally. Nuclear magnetic resonance (NMR) is uniquely equipped to provide structural information on IDRs at atomic resolution; however, existing NMR methods often pose a challenge for large molecular weight IDRs. Resonance assignment of IDRs using 15ND-detection was previously demonstrated and shown to overcome some of these limitations. Here, we improve the methodology by overcoming the need for deuterated buffers and provide better sensitivity and resolution at higher magnetic fields and physiological salt concentrations using transverse relaxation optimized spectroscopy (TROSY). Finally, large disordered regions with low sequence complexity can be assigned efficiently using these new methods as demonstrated by achieving near complete assignment of the 398-residue N-terminal IDR of the transcription factor NFAT1 harboring 18% prolines.

DOI: https://doi.org/10.1039/d2cc02005j
Front Mol Biosci. 2022 ;9 910956

Integrative Conformational Ensembles of Sic1 Using Different Initial Pools and Optimization Methods.

Gregory-Neal W Gomes, Ashley Namini, Claudiu C Gradinaru.

  Intrinsically disordered proteins play key roles in regulatory protein interactions, but their detailed structural characterization remains challenging. Here we calculate and compare conformational ensembles for the disordered protein Sic1 from yeast, starting from initial ensembles that were generated either by statistical sampling of the conformational landscape, or by molecular dynamics simulations. Two popular, yet contrasting optimization methods were used, ENSEMBLE and Bayesian Maximum Entropy, to achieve agreement with experimental data from nuclear magnetic resonance, small-angle X-ray scattering and single-molecule Förster resonance energy transfer. The comparative analysis of the optimized ensembles, including secondary structure propensity, inter-residue contact maps, and the distributions of hydrogen bond and pi interactions, revealed the importance of the physics-based generation of initial ensembles. The analysis also provides insights into designing new experiments that report on the least restrained features among the optimized ensembles. Overall, differences between ensembles optimized from different priors were greater than when using the same prior with different optimization methods. Generating increasingly accurate, reliable and experimentally validated ensembles for disordered proteins is an important step towards a mechanistic understanding of their biological function and involvement in various diseases.

Keywords:  IDP ensembles; NMR; SAXS; contact maps; molecular dynamics; smFRET

DOI:  https://doi.org/10.3389/fmolb.2022.910956
Biochim Biophys Acta Proteins Proteom. 2022 Aug 01. pii: S1570-9639(22)00075-9. [Epub ahead of print] 140828

Native mass spectrometry for the investigation of protein structural (dis)order.

Carlo Santambrogio, Erika Ponzini, Rita Grandori.

  A central challenge in structural biology is represented by dynamic and heterogeneous systems, as typically represented by proteins in solution, with the extreme case of intrinsically disordered proteins (IDPs) [1-3]. These proteins lack a specific threedimensional structure and have poorly organized secondary structure. For these reasons, they escape structural characterization by conventional biophysical methods. The investigation of these systems requires description of conformational ensembles, rather than of unique, defined structures or bundles of largely superimposable structures. Mass spectrometry (MS) has become a central tool in this field, offering a variety of complementary approaches to generate structural information on either folded or disordered proteins [4-6]. Two main categories of methods can be recognized. On one side, conformation-dependent reactions (such as cross-linking, covalent labeling, H/D exchange) are exploited to label molecules in solution, followed by the characterization of the labeling products by denaturing MS [7-11]. On the other side, non-denaturing ("native") MS can be used to directly explore the different conformational components in terms of geometry and structural compactness [12-16]. All these approaches have in common the capability to conjugate protein structure investigation with the peculiar analytical power of MS measurements, offering the possibility of assessing species distributions for folding and binding equilibria and the combination of both. These methods can be combined with characterization of noncovalent complexes [17, 18] and post-translational modifications [19-23]. This review focuses on the application of native MS to protein structure and dynamics investigation, with a general methodological section, followed by examples on specific proteins from our laboratory.

Keywords:  Intrinsically disordered proteins; Ion-mobility mass spectrometry; Native mass spectrometry; Protein conformational ensembles; Protein non-covalent complexes; Protein-ligand interactions; Protein-protein interactions

DOI:  https://doi.org/10.1016/j.bbapap.2022.140828
Biochemistry. 2022 Aug 02.

Protein Condensate Formation via Controlled Multimerization of Intrinsically Disordered Sequences.

Mikael V Garabedian, Zhihui Su, Jorge Dabdoub, Michelle Tong, Alexander Deiters, Daniel A Hammer, Matthew C Good.

Many proteins harboring low complexity or intrinsically disordered sequences (IDRs) are capable of undergoing liquid-liquid phase separation to form mesoscale condensates that function as biochemical niches with the ability to concentrate or sequester macromolecules and regulate cellular activity. Engineered disordered proteins have been used to generate programmable synthetic membraneless organelles in cells. Phase separation is governed by the strength of interactions among polypeptides with multivalency enhancing phase separation at lower concentrations. Previously, we and others demonstrated enzymatic control of IDR valency from multivalent precursors to dissolve condensed phases. Here, we develop noncovalent strategies to multimerize an individual IDR, the RGG domain of LAF-1, using protein interaction domains to regulate condensate formation in vitro and in living cells. First, we characterize modular dimerization of RGG domains at either terminus using cognate high-affinity coiled-coil pairs to form stable condensates in vitro. Second, we demonstrate temporal control over phase separation of RGG domains fused to FRB and FKBP in the presence of dimerizer. Further, using a photocaged dimerizer, we achieve optically induced condensation both in cell-sized emulsions and within live cells. Collectively, these modular tools allow multiple strategies to promote phase separation of a common core IDR for tunable control of condensate assembly.

DOI: https://doi.org/10.1021/acs.biochem.2c00250
Int J Biol Macromol. 2022 Aug 02. pii: S0141-8130(22)01642-7. [Epub ahead of print]

Intrinsically disordered BMP4 morphogen and the beak of the finch: Co-option of an ancient axial patterning system.

Prakash Kulkarni, Atish Mohanty, Ravi Salgia, Vladimir N Uversky.

  Darwin's finches, with the primary diversity in the shape and size of their beaks, represent an excellent model system to study speciation and adaptive evolution. It is generally held that evolution depends on the natural selection of heritable phenotypic variations originating from the genetic mutations. However, it is now increasingly evident that epigenetic transgenerational inheritance of phenotypic variation can also guide evolutionary change. Several studies have shown that the bone morphogenetic protein BMP4 is a major driver of beak morphology. A recent study explored variability of the morphological, genetic, and epigenetic differences in the adjacent "urban" and "rural" populations of two species of ground Darwin's finches on the Galápagos Islands and revealed significant changes in methylation patterns in several genes including those involved in the BMP/TGFß pathway in the sperm DNA compared to erythrocyte DNA. These observations indicated that epigenetic changes caused by environmental fluctuations can be passed on to the offspring. Nonetheless, the mechanism by which dysregulated expression of BMP4 impacts beak morphology remains poorly understood. Here, we show that BMP4 is an intrinsically disordered protein and present a causal a link between epigenetic changes, BMP4 dysregulation and the evolution of the beak of the finch by natural selection.

Keywords:  Beak; Bone morphogenetic protein 4; Co-option; Darwin's finches; Evolution; Intrinsically disordered protein

DOI:  https://doi.org/10.1016/j.ijbiomac.2022.07.203
Phys Chem Chem Phys. 2022 Aug 01.

Molecular dynamics simulations of an α-synuclein NAC domain fragment with a ff14IDPSFF IDP-specific force field suggest β-sheet intermediate states of fibrillation.

Cristian Privat, Sergio Madurga, Francesc Mas, Jaime Rubio-Martinez.

For the discovery of treatments against synucleinopathies, it is necessary to unravel and fully understand the mechanism of fibrillation of proteins involved. Among them, α-synuclein (αS) plays a key role in the development of these diseases through its aggregation into oligomers found in Lewy bodies. However, its structural disorder as an intrinsically disordered protein (IDP) makes its characterization by experimental techniques arduously difficult. Atomistic simulations aim to provide insights into this blank canvas and, fortunately, some studies have already suggested promising mechanisms. Still, it is urgent to consider the IDP features in simulations, so recently a lot of force fields designed to deal with IDPs have been developed. In this study, we have carried out a total of 12 μs simulations of an αS core fragment using a popular ff14SB AMBER force field and the ff14IDPSFF variation that includes a grid-based energy correction map (CMAP) method. The predicted chemical shifts from the simulations and those measured from the αS protein in the NMR solution indicate that ff14IDPSFF reproduces the experimental data more accurately. Moreover, structural analysis exhibits opposite trends between secondary structure propensities. The ff14SB force field preserves the α-helices found in the micelle-bound αS structure, which is used as an initial conformation, while ff14IDPSFF stands out with increased structural disorder and the formation of β-sheets, which suggests that the IDP-specific force field can capture more suitable conformations representing the possible intermediate states of the fibrillation process.

DOI: https://doi.org/10.1039/d2cp02042d
Comput Biol Chem. 2022 Jul 19. pii: S1476-9271(22)00116-5. [Epub ahead of print]100 107736

Remote communication between unstructured and structured regions of Bcl-2 tunes its ligand binding capacity: Mechanistic insights.

Debarati Paul, Premananda Basak, Shubhra Ghosh Dastidar.

  Bcl-2, the prototypic, anti-apoptotic member of Bcl-2 family possesses a long Intrinsically Disordered Region (IDR) of more than sixty amino acid residues. In spite of a number of experimental evidences on the influence of IDR to regulate the function of the protein, the molecular basis is not yet established. The present work with ~8µs conformational sampling of Bcl-2, using molecular dynamics in all atom description, offers a molecular mechanistic insight into the communication between the IDR and the structured region. The results indicate a highly significant role of the IDR in controlling the movements of the atoms which form the primary binding site. Although the influence of the IDR of the wild type Bcl-2 on its structured region, especially on the binding site, seems to be ordinary, but the hidden pathway of communication gets elucidated as the perturbation due to single-site phosphorylation on S70 works as a marker of the path; the contrast brought by the data obtained after truncating the IDR highlights it further. In wild type Bcl-2, apparently there is no direct communication between the IDR and binding site. But in the phosphorylated system, a communication channel between the IDR and the binding site has been established, as evidenced from the network analysis, which results in an increased correlation between the binding pocket residues and that redistributs the sampling of conformations of the system; one of the major consequences of these changes is witnessed in its enhanced affinity for binding with its partner Bax.

Keywords:  Bcl-2; Disordered region; Molecular dynamics; Phosphorylation; Population shift

DOI:  https://doi.org/10.1016/j.compbiolchem.2022.107736
J Chem Inf Model. 2022 Aug 03.

Accurate Description of Solvent-Exposed Salt Bridges with a Non-polarizable Force Field Incorporating Solvent Effects.

Han Liu, Haohao Fu, Christophe Chipot, Xueguang Shao, Wensheng Cai.

The strength of salt bridges resulting from the interaction of cations and anions is modulated by their environment. However, polarization of the solvent molecules by the charged moieties makes the accurate description of cation-anion interactions in an aqueous solution by means of a pairwise additive potential energy function and classical combination rules particularly challenging. In this contribution, aiming at improving the representation of solvent-exposed salt-bridge interactions with an all-atom non-polarizable force field, we put forth here a parametrization strategy. First, the interaction of a cation and an anion is characterized by hybrid quantum mechanical/molecular mechanics (QM/MM) potential of mean force (PMF) calculations, whereby constantly exchanging solvent molecules around the ions are treated at the quantum mechanical level. The Lennard-Jones (LJ) parameters describing the salt-bridge ion pairs are then optimized to match the reference QM/MM PMFs through the so-called nonbonded FIX, or NBFIX, feature of the CHARMM force field. We apply the new set of parameters, coined CHARMM36m-SBFIX, to the calculation of association constants for the ammonium-acetate and guanidinium-acetate complexes, the osmotic pressures for glycine zwitterions, guanidinium, and acetate ions, and to the simulation of both folded and intrinsically disordered proteins. Our findings indicate that CHARMM36m-SBFIX improves the description of solvent-exposed salt-bridge interactions, both structurally and thermodynamically. However, application of this force field to the standard binding free-energy calculation of a protein-ligand complex featuring solvent-excluded salt-bridge interactions leads to a poor reproduction of the experimental value, suggesting that the parameters optimized in an aqueous solution cannot be readily transferred to describe solvent-excluded salt-bridge interactions. Put together, owing to their sensitivity to the environment, modeling salt-bridge interactions by means of a single, universal set of LJ parameters remains a daunting theoretical challenge.

DOI: https://doi.org/10.1021/acs.jcim.2c00678
PLoS One. 2022 ;17(8): e0267370

Structural and biophysical properties of FopA, a major outer membrane protein of Francisella tularensis.

Nirupa Nagaratnam, Jose M Martin-Garcia, Jay-How Yang, Matthew R Goode, Gihan Ketawala, Felicia M Craciunescu, James D Zook, Manashi Sonowal, Dewight Williams, Thomas D Grant, Raimund Fromme, Debra T Hansen, Petra Fromme.

Francisella tularensis is an extremely infectious pathogen and a category A bioterrorism agent. It causes the highly contagious zoonosis, Tularemia. Currently, FDA approved vaccines against tularemia are unavailable. F. tularensis outer membrane protein A (FopA) is a well-studied virulence determinant and protective antigen against tularemia. It is a major outer membrane protein (Omp) of F. tularensis. However, FopA-based therapeutic intervention is hindered due to lack of complete structural information for membrane localized mature FopA. In our study, we established recombinant expression, monodisperse purification, crystallization and X-ray diffraction (~6.5 Å) of membrane localized mature FopA. Further, we performed bioinformatics and biophysical experiments to unveil its structural organization in the outer membrane. FopA consists of 393 amino acids and has less than 40% sequence identity to known bacterial Omps. Using comprehensive sequence alignments and structure predictions together with existing partial structural information, we propose a two-domain organization for FopA. Circular dichroism spectroscopy and heat modifiability assay confirmed FopA has a β-barrel domain consistent with alphafold2's prediction of an eight stranded β-barrel at the N-terminus. Small angle X-ray scattering (SAXS) and native-polyacrylamide gel electrophoresis revealed FopA purified in detergent micelles is predominantly dimeric. Molecular density derived from SAXS at 31 Å shows putative dimeric N-terminal β-barrels surrounded by detergent corona and connected to C-terminal domains via flexible linker. Disorder analysis predicts N- and C-terminal domains are interspersed by a long intrinsically disordered region and alphafold2 predicts this region to be largely unstructured. Taken together, we propose a dimeric, two-domain organization of FopA in the outer membrane: the N-terminal β-barrel is membrane embedded, provides dimerization interface and tethers to membrane extrinsic C-terminal domain via long flexible linker. Structure determination of membrane localized mature FopA is essential to understand its role in pathogenesis and develop anti-tularemia therapeutics. Our results pave the way towards it.

DOI: https://doi.org/10.1371/journal.pone.0267370
Anal Biochem. 2022 Aug 02. pii: S0003-2697(22)00288-3. [Epub ahead of print] 114828

Review: Investigating the aggregation of amyloid beta with surface plasmon resonance: Do different approaches yield different results?

Kimberly A Young, Ricardo L Mancera.

  Aggregation of amyloid beta into amyloid plaques in the brain is a hallmark characteristic of Alzheimer's disease. Therapeutics aimed at preventing or retarding amyloid formation often rely on detailed characterization of the underlying mechanism and kinetics of protein aggregation. Surface plasmon resonance (SPR) spectroscopy is a robust technique used to determine binding affinity and kinetics of biomolecular interactions. This approach has been used to characterize the mechanism of aggregation of amyloid beta but there are multiple pitfalls that need to be addressed when working with this and other amyloidogenic proteins. The choice of method for analyte preparation and ligand immobilization to a sensor chip can lead to different theoretical and practical implications in terms of the mathematical modelling of binding data, different mechanisms of binding and the presence of different interacting species. This review examines preparation methods for SPR characterisation of the aggregation of amyloid beta and their influence on the findings derived from such studies.

Keywords:  Aggregation; Amyloid-beta; Amyloidogenic; Intrinsically disordered protein; Neurodegenerative disease; Surface plasmon resonance

DOI:  https://doi.org/10.1016/j.ab.2022.114828
Sci Adv. 2022 Aug 05. 8(31): eabm7528

Lipid-driven condensation and interfacial ordering of FUS.

Sayantan Chatterjee, Daria Maltseva, Yelena Kan, Elnaz Hosseini, Grazia Gonella, Mischa Bonn, Sapun H Parekh.

Protein condensation into liquid-like structures is critical for cellular compartmentalization, RNA processing, and stress response. Research on protein condensation has primarily focused on membraneless organelles in the absence of lipids. However, the cellular cytoplasm is full of lipid interfaces, yet comparatively little is known about how lipids affect protein condensation. Here, we show that nonspecific interactions between lipids and the disordered fused in sarcoma low-complexity (FUS LC) domain strongly affect protein condensation. In the presence of anionic lipids, FUS LC formed lipid-protein clusters at concentrations more than 30-fold lower than required for pure FUS LC. Lipid-triggered FUS LC clusters showed less dynamic protein organization than canonical, lipid-free FUS LC condensates. Lastly, we found that phosphatidylserine membranes promoted FUS LC condensates having β sheet structures, while phosphatidylglycerol membranes initiated unstructured condensates. Our results show that lipids strongly influence FUS LC condensation, suggesting that protein-lipid interactions modulate condensate formation in cells.

DOI: https://doi.org/10.1126/sciadv.abm7528
Biochim Biophys Acta Proteins Proteom. 2022 Aug 01. pii: S1570-9639(22)00073-5. [Epub ahead of print] 140826

Designer D-peptides targeting the N-terminal region of α-synuclein to prevent parkinsonian-associated fibrilization and cytotoxicity.

John R Horsley, Blagojce Jovcevski, Tara L Pukala, Andrew D Abell.

  The deposition of α-synuclein (αS) aggregates in the gut and the brain is ever present in cases of Parkinson's disease. While the central non-amyloidogenic-component (NAC) region of αS plays a critical role in fibrilization, recent studies have identified a specific sequence from within the N-terminal region (NTR, residues 36-42) as a key modulator of αS fibrilization. Due to the lack of effective therapeutics which specifically target αS aggregates, we have developed a strategy to prevent the aggregation and subsequent toxicity attributed to αS fibrilization utilizing NTR targeting peptides. In this study, L- and D-isoforms of a hexa- (VAQKTV-Aib, 77-82 NAC) and heptapeptide (GVLYVGS-Aib, 36-42 NTR) containing a self-recognition component unique to αS, as well as a C-terminal disruption element, were synthesized to target primary sequence regions of αS that modulate fibrilization. The D-peptide that targets the NTR (NTR-TP-D) was shown by ThT fluorescence assays and TEM to be the most effective at preventing fibril formation and elongation, as well as increasing the abundance of soluble monomeric αS. In addition, NTR-TP-D alters the conformation of destabilised monomers into a less aggregation-prone state and reduces the hydrophobicity of αS fibrils via fibril remodelling. Furthermore, both NTR-TP isoforms alleviate the cytotoxic effects of αS aggregates in both Neuro-2a and Caco-2 cells. Together, this study highlights how targeting the NTR of αS using D-isoform peptide inhibitors may effectively combat the deleterious effects of αS fibrilization and paves the way for future drug design to utilise such an approach to treat Parkinson's disease.

Keywords:  Amyloid fibrils; Drug design; Peptide inhibitors; Protein aggregation; α-Synuclein

DOI:  https://doi.org/10.1016/j.bbapap.2022.140826
Front Mol Biosci. 2022 ;9 960806

Regulation of TIA-1 Condensates: Zn2+ and RGG Motifs Promote Nucleic Acid Driven LLPS and Inhibit Irreversible Aggregation.

Danella L West, Fionna E Loughlin, Francisco Rivero-Rodríguez, Naveen Vankadari, Alejandro Velázquez-Cruz, Laura Corrales-Guerrero, Irene Díaz-Moreno, Jacqueline A Wilce.

  Stress granules are non-membrane bound RNA-protein granules essential for survival during acute cellular stress. TIA-1 is a key protein in the formation of stress granules that undergoes liquid-liquid phase separation by association with specific RNAs and protein-protein interactions. However, the fundamental properties of the TIA-1 protein that enable phase-separation also render TIA-1 susceptible to the formation of irreversible fibrillar aggregates. Despite this, within physiological stress granules, TIA-1 is not present as fibrils, pointing to additional factors within the cell that prevent TIA-1 aggregation. Here we show that heterotypic interactions with stress granule co-factors Zn2+ and RGG-rich regions from FUS each act together with nucleic acid to induce the liquid-liquid phase separation of TIA-1. In contrast, these co-factors do not enhance nucleic acid induced fibril formation of TIA-1, but rather robustly inhibit the process. NMR titration experiments revealed specific interactions between Zn2+ and H94 and H96 in RRM2 of TIA-1. Strikingly, this interaction promotes multimerization of TIA-1 independently of the prion-like domain. Thus, through different molecular mechanisms, these stress granule co-factors promote TIA-1 liquid-liquid phase separation and suppress fibrillar aggregates, potentially contributing to the dynamic nature of stress granules and the cellular protection that they provide.

Keywords:  RGG motif; RNA binding protein; RRM; TIA1; amyloid fibril; liquid-liquid phase separation; prion-like domain; zinc

DOI:  https://doi.org/10.3389/fmolb.2022.960806