bims-indpro 2022-11-20 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022‒11‒20
twenty papers selected by
Sara Mingu
Johannes Gutenberg University

Shapeshifting tau: from intrinsically disordered to paired-helical filaments.
Intrinsically disordered interaction network in an RNA chaperone revealed by native mass spectrometry.
Concerted enhanced-sampling simulations to elucidate the helix-fibril transition pathway of intrinsically disordered α-Synuclein.
Regulation of RB1CC1/FIP200 stability and autophagy function by CREBBP-mediated acetylation in an intrinsically disordered region.
In Vitro Amplification of Pathogenic Tau Seeds from Neurodegenerative Disease Patient Brains.
Combining enhanced sampling and deep learning dimensionality reduction for the study of the heat shock protein B8 and its pathological mutant K141E.
Mapping Interactions of the Intrinsically Disordered C-Terminal Regions of Tetrameric p53 by Segmental Isotope Labeling and NMR.
Molecular dynamics simulations of the adsorption of an intrinsically disordered protein: Force field and water model evaluation in comparison with experiments.
Ubiquitin assisted phase separation of Dishevelled-2 promotes Wnt signaling.
The evolution and polymorphism of mono-amino acid repeats in androgen receptor and their regulatory role in health and disease.
Condensation of SEUSS promotes hyperosmotic stress tolerance in Arabidopsis.
Pure shift amide detection in conventional and TROSY-type experiments of 13C,15N-labeled proteins.
A glutamine-based single α-helix scaffold to target globular proteins.
Improving IDP theoretical chemical shift accuracy and efficiency through a combined MD/ADMA/DFT and machine learning approach.
Structural insights into the mechanism of HIV-1 Tat secretion from the plasma membrane.
Phase separation and other forms of α-Synuclein self-assemblies.
Acetylation and phosphorylation processes modulate Tau's binding to microtubules: A molecular dynamics study.
Sequence Context and Complex Hofmeister Salt Interactions Dictate Phase Separation Propensity of Resilin-like Polypeptides.
Binding of two zinc ions promotes liquid-liquid phase separation of Tau.
Soft disorder modulates the assembly path of protein complexes.

Essays Biochem. 2022 Nov 14. pii: EBC20220150. [Epub ahead of print]

Shapeshifting tau: from intrinsically disordered to paired-helical filaments.

Kurtis Mengham, Youssra Al-Hilaly, Sebastian Oakley, Kamillia Kasbi, Mahmoud B Maina, Louise C Serpell.

  Tau is an intrinsically disordered protein that has the ability to self-assemble to form paired helical and straight filaments in Alzheimer's disease, as well as the ability to form additional distinct tau filaments in other tauopathies. In the presence of microtubules, tau forms an elongated form associated with tubulin dimers via a series of imperfect repeats known as the microtubule binding repeats. Tau has recently been identified to have the ability to phase separate in vitro and in cells. The ability of tau to adopt a wide variety of conformations appears fundamental both to its biological function and also its association with neurodegenerative diseases. The recently highlighted involvement of low-complexity domains in liquid-liquid phase separation provides a critical link between the soluble function and the insoluble dysfunctional properties of tau.

Keywords:  Alzheimer's disease; amyloid; intrinsically disordered proteins; liquid liquid phase separation; protein misfolding; tau

DOI:  https://doi.org/10.1042/EBC20220150
Proc Natl Acad Sci U S A. 2022 Nov 22. 119(47): e2208780119

Intrinsically disordered interaction network in an RNA chaperone revealed by native mass spectrometry.

Samantha H Sarni, Jorjethe Roca, Chen Du, Mengxuan Jia, Hantian Li, Ana Damjanovic, Ewelina M Małecka, Vicki H Wysocki, Sarah A Woodson.

  RNA-binding proteins contain intrinsically disordered regions whose functions in RNA recognition are poorly understood. The RNA chaperone Hfq is a homohexamer that contains six flexible C-terminal domains (CTDs). The effect of the CTDs on Hfq's integrity and RNA binding has been challenging to study because of their sequence identity and inherent disorder. We used native mass spectrometry coupled with surface-induced dissociation and molecular dynamics simulations to disentangle the arrangement of the CTDs and their impact on the stability of Escherichia coli Hfq with and without RNA. The results show that the CTDs stabilize the Hfq hexamer through multiple interactions with the core and between CTDs. RNA binding perturbs this network of CTD interactions, destabilizing the Hfq ring. This destabilization is partially compensated by binding of RNAs that contact multiple surfaces of Hfq. By contrast, binding of short RNAs that only contact one or two subunits results in net destabilization of the complex. Together, the results show that a network of intrinsically disordered interactions integrate RNA contacts with the six subunits of Hfq. We propose that this CTD network raises the selectivity of RNA binding.

Keywords:  RNA chaperone; intrinsically disordered protein; ion mobility mass spectrometry; small RNA; surface-induced ion dissociation

DOI:  https://doi.org/10.1073/pnas.2208780119
Int J Biol Macromol. 2022 Nov 12. pii: S0141-8130(22)02637-X. [Epub ahead of print]

Concerted enhanced-sampling simulations to elucidate the helix-fibril transition pathway of intrinsically disordered α-Synuclein.

Archi Saurabh, N Prakash Prabhu.

  Fibril formation of α-synuclein is linked with Parkinson's disease. The intrinsically disordered nature of α-syn provides extensive conformational plasticity and becomes difficult to characterize its transition pathway from native monomeric to disease-associated fibril form. We implemented different simulation methods such as steered dynamics-umbrella sampling, and replica-exchange and conventional MD simulations to access various conformational states of α-syn. Nineteen distinct intermediate structures were identified by free energy landscape and cluster analysis. They were then sorted based on secondary structure and solvent exposure of fibril-core residues to illustrate the fibril dissociation pathway. The analysis showed that following the initial dissociation of the polypeptide chain from the fibril, α-syn might form either compact-conformations by long-range interactions or extended-conformations stabilized by local interactions. This leads α-syn to adapt two different pathways. The secondary structure, solvation, contact distance, interaction energies and backbone dihedrals of thirty-two selected residues were analyzed for all the 19 intermediates. The results suggested that formation of β-turns, reorganization of salt bridges, and dihedral changes in the hydrophobic regions are the major driving forces for helix-fibril transition. Structural features of the intermediates also correlated with the earlier experimental and computational studies. The study provides critical information on the fibrillation pathway of α-syn.

Keywords:  Intrinsically disordered protein; Molecular dynamics simulation; α-Synuclein fibril pathway

DOI:  https://doi.org/10.1016/j.ijbiomac.2022.11.079
Autophagy. 2022 Nov 17.

Regulation of RB1CC1/FIP200 stability and autophagy function by CREBBP-mediated acetylation in an intrinsically disordered region.

Fei Yi, Chunmiao Cai, Banzhan Ruan, Mingang Hao, Syn Kok Yeo, Michael Haas, Fuchun Yang, Xiaoting Zhang, Jun-Lin Guan.

  RB1CC1/FIP200 is an essential macroautophagy/autophagy protein that plays an important role in a variety of biological and disease processes through its canonical autophagy-dependent and -independent functions. However, it remains largely unknown whether post-translational modifications could regulate RB1CC1 and its associated autophagy functions. Here, we report acetylation of several lysine residues of RB1CC1 by acetyltransferase CREBBP (CREB binding protein), with K276 as the major CREBBP acetylation site. K276 is also identified as a ubiquitination site by mass spectrometry, and acetylation at this site reduces ubiquitination of RB1CC1 to inhibit its ubiquitin-dependent degradation. We also find that RB1CC1 contains an N-terminal intrinsically disordered region (IDR) capable of forming liquid-liquid phase separation (LLPS) in vitro, which may drive formation of RB1CC1 puncta with LLPS properties in cells independent of SQSTM1/p62 and other autophagy receptors CALCOCO2/NDP52, NBR1, TAX1BP1 and OPTN. Mutational analysis shows that both K276 acetylation and the N-terminal IDR containing it are important for maintaining canonical autophagy function of RB1CC1 in breast cancer cells. Our findings demonstrate regulation of RB1CC1 by a new post-translational mechanism and suggest potential therapeutic application of inducing RB1CC1 degradation through blocking K276 acetylation in the treatment of cancer and other diseases.

Keywords:  CREBBP; RB1CC1; autophagy; liquid-liquid phase separation; protein acetylation

DOI:  https://doi.org/10.1080/15548627.2022.2148432
Methods Mol Biol. 2023 ;2561 279-292

In Vitro Amplification of Pathogenic Tau Seeds from Neurodegenerative Disease Patient Brains.

Hong Xu, Virginia M-Y Lee.

  Aggregated microtubule-associated protein tau (tau) is the hallmark lesion of a group of neurodegenerative diseases, termed tauopathies. Normal endogenous tau is highly soluble and intrinsically disordered when it is not bound to microtubules. Pathological tau proteins are aggregates of bioactive filaments capable of inducing their normal counterparts into pathological conformations that are human tauopathy dependent. Taking advantage of this feature, we established an in vitro seeding reaction to amplify faithfully human-derived tau strains. This approach allows us to expand the quantity and improve the quality of pathogenic tau strains derived from human patient postmortem brains and to further understand tau pathogenesis in models of tauopathy. Here, we describe the approach to generate human pathogenic tau using human-derived tau seeds and recombinant human tau in vitro.

Keywords:  Amplification; Cell free; In vitro seeding; Tau spreading; Tau strains; Tauopathy

DOI:  https://doi.org/10.1007/978-1-0716-2655-9_15
RSC Adv. 2022 Nov 03. 12(49): 31996-32011

Combining enhanced sampling and deep learning dimensionality reduction for the study of the heat shock protein B8 and its pathological mutant K141E.

Daniele Montepietra, Ciro Cecconi, Giorgia Brancolini.

The biological functions of proteins closely depend on their conformational dynamics. This aspect is especially relevant for intrinsically disordered proteins (IDP) for which structural ensembles often offer more useful representations than individual conformations. Here we employ extensive enhanced sampling temperature replica-exchange atomistic simulations (TREMD) and deep learning dimensionality reduction to study the conformational ensembles of the human heat shock protein B8 and its pathological mutant K141E, for which no experimental 3D structures are available. First, we combined homology modelling with TREMD to generate high-dimensional data sets of 3D structures. Then, we employed a recently developed machine learning based post-processing algorithm, EncoderMap, to project the large conformational data sets into meaningful two-dimensional maps that helped us interpret the data and extract the most significant conformations adopted by both proteins during TREMD. These studies provide the first 3D structural characterization of HSPB8 and reveal the effects of the pathogenic K141E mutation on its conformational ensembles. In particular, this missense mutation appears to increase the compactness of the protein and its structural variability, at the same time rearranging the hydrophobic patches exposed on the protein surface. These results offer the possibility of rationalizing the pathogenic effects of the K141E mutation in terms of conformational changes.

DOI: https://doi.org/10.1039/d2ra04913a
Biochemistry. 2022 Nov 15.

Mapping Interactions of the Intrinsically Disordered C-Terminal Regions of Tetrameric p53 by Segmental Isotope Labeling and NMR.

Alexander S Krois, Sangho Park, Maria A Martinez-Yamout, H Jane Dyson, Peter E Wright.

The C-terminal region of the tumor suppressor protein p53 contains three domains, nuclear localization signal (NLS), tetramerization domain (TET), and C-terminal regulatory domain (CTD), which are essential for p53 function. Characterization of the structure and interactions of these domains within full-length p53 has been limited by the overall size and flexibility of the p53 tetramer. Using trans-intein splicing, we have generated full-length p53 constructs in which the C-terminal region is isotopically labeled with 15N for NMR analysis, allowing us to obtain atomic-level information on the C-terminal domains in the context of the full-length protein. Resonances of NLS and CTD residues have narrow linewidths, showing that these regions are largely solvent-exposed and dynamically disordered, whereas resonances from the folded TET are broadened beyond detection. Two regions of the CTD, spanning residues 369-374 and 381-388 and with high lysine content, make dynamic and sequence-independent interactions with DNA in regions that flank the p53 recognition element. The population of DNA-bound states increases as the length of the flanking regions is extended up to approximately 20 base pairs on either side of the recognition element. Acetylation of K372, K373, and K382, using a construct of the transcriptional coactivator CBP containing the TAZ2 and acetyltransferase domains, inhibits interaction of the CTD with DNA. This work provides high-resolution insights into the behavior of the intrinsically disordered C-terminal regions of p53 within the full-length tetramer and the molecular basis by which the CTD mediates DNA binding and specificity.

DOI: https://doi.org/10.1021/acs.biochem.2c00528
Front Mol Biosci. 2022 ;9 958175

Molecular dynamics simulations of the adsorption of an intrinsically disordered protein: Force field and water model evaluation in comparison with experiments.

Mona Koder Hamid, Linda K Månsson, Viktoriia Meklesh, Per Persson, Marie Skepö.

  This study investigates possible structural changes of an intrinsically disordered protein (IDP) when it adsorbs to a solid surface. Experiments on IDPs primarily result in ensemble averages due to their high dynamics. Therefore, molecular dynamics (MD) simulations are crucial for obtaining more detailed information on the atomistic and molecular levels. An evaluation of seven different force field and water model combinations have been applied: (A) CHARMM36IDPSFF + CHARMM-modified TIP3P, (B) CHARMM36IDPSFF + TIP4P-D, (C) CHARMM36m + CHARMM-modified TIP3P, (D) AMBER99SB-ILDN + TIP3P, (E) AMBER99SB-ILDN + TIP4P-D, (F) AMBERff03ws + TIP4P/2005, and (G) AMBER99SB-disp + disp-water. The results have been qualitatively compared with those of small-angle X-ray scattering, synchrotron radiation circular dichroism spectroscopy, and attenuated total reflectance Fourier transform infrared spectroscopy. The model IDP corresponds to the first 33 amino acids of the N-terminal of the magnesium transporter A (MgtA) and is denoted as KEIF. With a net charge of +3, KEIF is found to adsorb to the anionic synthetic clay mineral Laponite® due to the increase in entropy from the concomitant release of counterions from the surface. The experimental results show that the peptide is largely disordered with a random coil conformation, whereas the helical content (α- and/or 310-helices) increased upon adsorption. MD simulations corroborate these findings and further reveal an increase in polyproline II helices and an extension of the peptide conformation in the adsorbed state. In addition, the simulations provided atomistic resolution of the adsorbed ensemble of structures, where the arginine residues had a high propensity to form hydrogen bonds with the surface. Simulations B, E, and G showed significantly better agreement with experiments than the other simulations. Particularly noteworthy is the discovery that B and E with TIP4P-D water had superior performance to their corresponding simulations A and D with TIP3P-type water. Thus, this study shows the importance of the water model when simulating IDPs and has also provided an insight into the structural changes of surface-active IDPs induced by adsorption, which may play an important role in their function.

Keywords:  adsorption; conformational ensemble; force field (FF); intrinsically disordered proteins (IDPs); molecular dynamics; water models

DOI:  https://doi.org/10.3389/fmolb.2022.958175
J Cell Sci. 2022 Nov 18. pii: jcs.260284. [Epub ahead of print]

Ubiquitin assisted phase separation of Dishevelled-2 promotes Wnt signaling.

Vaishna Vamadevan, Neelam Chaudhary, Subbareddy Maddika.

  Dishvelled-2 (Dvl2) is an essential component of Wnt pathway, which controls several cell fate decisions during development such as proliferation, survival and differentiation. Dvl2 forms higher-order protein assemblies in cell that are critical for relaying the signal from upstream Wnt ligand-Frizzled receptor binding to downstream effector β-catenin activation. However, the precise molecular nature and contribution of Dvl2 protein assemblies during Wnt signaling is unknown. Here, we show that Dvl2 forms protein condensates driven by liquid-liquid phase separation. An intrinsically disordered region (IDR) on the N-terminus is essential for Dvl2 phase separation. Importantly, we identified a HECT-E3 ligase WWP2 as an essential driver of Dvl2 phase separation in vitro and in cells. We demonstrated that ubiquitination of Dvl2 through K63 linkage by WWP2 is required for formation of Dvl2 condensates. Phase separated Dvl2 activates Wnt signaling by sequestering the components of destruction complex and thus relieving β-catenin. Together, our results reveal a ubiquitination-dependent liquid-liquid phase separation as a new identity for Dvl2 in cells, which is necessary for transduction of Wnt signaling.

Keywords:  Dishevelled; Phase separation; Protein condensates; Ubiquitin; WWP2; Wnt signaling

DOI:  https://doi.org/10.1242/jcs.260284
Front Med (Lausanne). 2022 ;9 1019803

The evolution and polymorphism of mono-amino acid repeats in androgen receptor and their regulatory role in health and disease.

Attila Meszaros, Junaid Ahmed, Giorgio Russo, Peter Tompa, Tamas Lazar.

  Androgen receptor (AR) is a key member of nuclear hormone receptors with the longest intrinsically disordered N-terminal domain (NTD) in its protein family. There are four mono-amino acid repeats (polyQ1, polyQ2, polyG, and polyP) located within its NTD, of which two are polymorphic (polyQ1 and polyG). The length of both polymorphic repeats shows clinically important correlations with disease, especially with cancer and neurodegenerative diseases, as shorter and longer alleles exhibit significant differences in expression, activity and solubility. Importantly, AR has also been shown to undergo condensation in the nucleus by liquid-liquid phase separation, a process highly sensitive to protein solubility and concentration. Nonetheless, in prostate cancer cells, AR variants also partition into transcriptional condensates, which have been shown to alter the expression of target gene products. In this review, we summarize current knowledge on the link between AR repeat polymorphisms and cancer types, including mechanistic explanations and models comprising the relationship between condensate formation, polyQ1 length and transcriptional activity. Moreover, we outline the evolutionary paths of these recently evolved amino acid repeats across mammalian species, and discuss new research directions with potential breakthroughs and controversies in the literature.

Keywords:  aggregation; amino acid repeats; androgen receptor; cancer; phase separation; phylogenetics; polyQ; polymorphism

DOI:  https://doi.org/10.3389/fmed.2022.1019803
Nat Chem Biol. 2022 Nov 14.

Condensation of SEUSS promotes hyperosmotic stress tolerance in Arabidopsis.

Boyu Wang, Honghong Zhang, Junling Huai, Fangyu Peng, Jie Wu, Rongcheng Lin, Xiaofeng Fang.

Osmotic stress imposed by drought and high salinity inhibits plant growth and crop yield. However, our current knowledge on the mechanism by which plants sense osmotic stress is still limited. Here, we identify the transcriptional regulator SEUSS (SEU) as a key player in hyperosmotic stress response in Arabidopsis. SEU rapidly coalesces into liquid-like nuclear condensates when extracellular osmolarity increases. The intrinsically disordered region 1 (IDR1) of SEU is responsible for its condensation. IDR1 undergoes conformational changes to adopt more compact states after an increase in molecular crowding both in vitro and in cells, and two predicted α-helical peptides are required. SEU condensation is indispensable for osmotic stress tolerance, and loss of SEU dramatically compromises the expression of stress tolerance genes. Our work uncovers a critical role of biomolecular condensates in cellular stress perception and response and expands our understanding of the osmotic stress pathway.

DOI: https://doi.org/10.1038/s41589-022-01196-z
J Biomol NMR. 2022 Nov 18.

Pure shift amide detection in conventional and TROSY-type experiments of 13C,15N-labeled proteins.

Jens D Haller, Andrea Bodor, Burkhard Luy.

  Large coupling networks in uniformly 13C,15N-labeled biomolecules induce broad multiplets that even in flexible proteins are frequently not recognized as such. The reason is that given multiplets typically consist of a large number of individual resonances that result in a single broad line, in which individual components are no longer resolved. We here introduce a real-time pure shift acquisition scheme for the detection of amide protons which is based on 13C-BIRDr,X. As a result the full homo- and heteronuclear coupling network can be suppressed at low power leading to real singlets at substantially improved resolution and uncompromised sensitivity. The method is tested on a small globular and an intrinsically disordered protein (IDP) where the average spectral resolution is increased by a factor of ~ 2 and higher. Equally important, the approach works without saturation of water magnetization for solvent suppression and exchanging amide protons are not affected by saturation transfer.

Keywords:  Amide detection; BIRD; High resolution; Homonuclear decoupling; IDPs; Proteins; Pure shift; Real-time; Solution state

DOI:  https://doi.org/10.1007/s10858-022-00406-z
Nat Commun. 2022 Nov 18. 13(1): 7073

A glutamine-based single α-helix scaffold to target globular proteins.

Albert Escobedo, Jonathan Piccirillo, Juan Aranda, Tammo Diercks, Borja Mateos, Carla Garcia-Cabau, Macarena Sánchez-Navarro, Busra Topal, Mateusz Biesaga, Lasse Staby, Birthe B Kragelund, Jesús García, Oscar Millet, Modesto Orozco, Murray Coles, Ramon Crehuet, Xavier Salvatella.

The binding of intrinsically disordered proteins to globular ones can require the folding of motifs into α-helices. These interactions offer opportunities for therapeutic intervention but their modulation with small molecules is challenging because they bury large surfaces. Linear peptides that display the residues that are key for binding can be targeted to globular proteins when they form stable helices, which in most cases requires their chemical modification. Here we present rules to design peptides that fold into single α-helices by instead concatenating glutamine side chain to main chain hydrogen bonds recently discovered in polyglutamine helices. The resulting peptides are uncharged, contain only natural amino acids, and their sequences can be optimized to interact with specific targets. Our results provide design rules to obtain single α-helices for a wide range of applications in protein engineering and drug design.

DOI: https://doi.org/10.1038/s41467-022-34793-6
Phys Chem Chem Phys. 2022 Nov 14.

Improving IDP theoretical chemical shift accuracy and efficiency through a combined MD/ADMA/DFT and machine learning approach.

Michael J Bakker, Arnošt Mládek, Hugo Semrád, Vojtěch Zapletal, Jana Pavlíková Přecechtělová.

This work extends the multi-scale computational scheme for the quantum mechanics (QM) calculations of Nuclear Magnetic Resonance (NMR) chemical shifts (CSs) in proteins that lack a well-defined 3D structure. The scheme couples the sampling of an intrinsically disordered protein (IDP) by classical molecular dynamics (MD) with protein fragmentation using the adjustable density matrix assembler (ADMA) and density functional theory (DFT) calculations. In contrast to our early investigation on IDPs (Pavlíková Přecechtělová et al., J. Chem. Theory Comput., 2019, 15, 5642-5658) and the state-of-the art NMR calculations for structured proteins, a partial re-optimization was implemented on the raw MD geometries in vibrational normal mode coordinates to enhance the accuracy of the MD/ADMA/DFT computational scheme. In addition, machine-learning based cluster analysis was performed on the scheme to explore its potential in producing protein structure ensembles (CLUSTER ensembles) that yield accurate CSs at a reduced computational cost. The performance of the cluster-based calculations is validated against results obtained with conventional structural ensembles consisting of MD snapshots extracted from the MD trajectory at regular time intervals (REGULAR ensembles). CS calculations performed with the refined MD/ADMA/DFT framework employed the 6-311++G(d,p) basis set that outperformed IGLO-III calculations with the same density functional approximation (B3LYP) and both explicit and implicit solvation. The partial geometry optimization did not universally improve the agreement of computed CSs with the experiment but substantially decreased errors associated with the ensemble averaging. A CLUSTER ensemble with 50 structures yielded ensemble averages close to those obtained with a REGULAR ensemble consisting of 500 MD frames. The cluster based calculations thus required only a fraction of the computational time.

DOI: https://doi.org/10.1039/d2cp01638a
J Mol Biol. 2022 Nov 09. pii: S0022-2836(22)00500-9. [Epub ahead of print] 167880

Structural insights into the mechanism of HIV-1 Tat secretion from the plasma membrane.

Ruba H Ghanam, Gunnar N Eastep, Jamil S Saad.

  Human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (Tat) is a small, intrinsically disordered basic protein that plays diverse roles in the HIV-1 replication cycle, including promotion of efficient viral RNA transcription. Tat is released by infected cells and subsequently absorbed by healthy cells, thereby contributing to HIV-1 pathogenesis including HIV-associated neurocognitive disorder. It has been shown that, in HIV-1-infected primary CD4 T-cells, Tat accumulates at the plasma membrane (PM) for secretion, a mechanism mediated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, the structural basis for Tat interaction with the PM and thereby secretion is lacking. Herein, we employed NMR and biophysical methods to characterize Tat86 (86 amino acids) interactions with PI(4,5)P2 and lipid nanodiscs (NDs). Our data revealed that Arg49, Lys50 and Lys51 (RKK motif) constitute the PI(4,5)P2 binding site, that Tat86 interaction with lipid NDs is dependent on PI(4,5)P2 and phosphatidylserine (PS), and that the arginine-rich motif (RRQRRR) preferentially interacts with PS. Furthermore, we show that Trp11, previously implicated in Tat secretion, penetrates deeply in the membrane; substitution of Trp11 severely reduced Tat86 interaction with membranes. Deletion of the entire highly basic region and Trp11 completely abolished Tat86 binding to membrane. Our data support a mechanism by which HIV-1 Tat secretion from the PM is mediated by a tripartite signal consisting of binding of the RKK motif to PI(4,5)P2, arginine-rich motif to PS, and penetration of Trp11 in the membrane. Altogether, these findings provide new insights into the molecular requirements for Tat binding to membranes during secretion. Running Title: Determinants of HIV-1 Tat-membrane interactions.

Keywords:  5)P(2); HIV-1; PI(4; Tat; membrane; secretion

DOI:  https://doi.org/10.1016/j.jmb.2022.167880
Essays Biochem. 2022 Nov 14. pii: EBC20220055. [Epub ahead of print]

Phase separation and other forms of α-Synuclein self-assemblies.

Manisha Poudyal, Arunima Sakunthala, Semanti Mukherjee, Laxmikant Gadhe, Samir K Maji.

  α-Synuclein (α-Syn) is a natively unstructured protein, which self-assembles into higher-order aggregates possessing serious pathophysiological implications. α-Syn aberrantly self-assembles into protein aggregates, which have been widely implicated in Parkinson's disease (PD) pathogenesis and other synucleinopathies. The self-assembly of α-Syn involves the structural conversion of soluble monomeric protein into oligomeric intermediates and eventually fibrillar aggregates of amyloids with cross-β-sheet rich conformation. These aggregated α-Syn species majorly constitute the intraneuronal inclusions, which is a hallmark of PD neuropathology. Self-assembly/aggregation of α-Syn is not a single-state conversion process as unfolded protein can access multiple conformational states through the formation of metastable, transient pre-fibrillar intermediate species. Recent studies have indicated that soluble oligomers are the potential neurotoxic species responsible for cell death in PD pathogenesis. The heterogeneous and transient nature of oligomers formed during the early stage of aggregation pathway limit their detailed study in understanding the structure-toxicity relationship. Moreover, the precise molecular events occurring in the early stage of α-Syn aggregation process majorly remain unsolved. Recently, liquid-liquid phase separation (LLPS) of α-Syn has been designated as an alternate nucleation mechanism, which occurs in the early lag phase of the aggregation pathway leading to the formation of dynamic supramolecular assemblies. The stronger self-association among the protein molecules triggers the irreversible liquid-to-solid transition of these supramolecular assemblies into the amyloid-like hydrogel, which may serve as a reservoir entrapping toxic oligomeric intermediates and fibrils. This review strives to provide insights into different modes of α-Syn self-assemblies including LLPS-mediated self-assembly and its recent advancements.

Keywords:  Fibrils; Phase separation; alpha synuclein; amyloid; oligomerization; protein aggregation

DOI:  https://doi.org/10.1042/EBC20220055
Biochim Biophys Acta Gen Subj. 2022 Nov 11. pii: S0304-4165(22)00194-5. [Epub ahead of print]1867(2): 130276

Acetylation and phosphorylation processes modulate Tau's binding to microtubules: A molecular dynamics study.

Tarsila G Castro, Tiago Ferreira, Teresa Matamá, Florentina-Daniela Munteanu, Artur Cavaco-Paulo.

  The microtubule-associated protein Tau has its normal function impaired when undergoing post-translational modifications. In this work, molecular modelling techniques were used to infer the effects of acetylation and phosphorylation in Tau's overall conformation, electrostatics, and interactions, but mostly in Tau's ability to bind microtubules. Reported harmful Lys sites were mutated by its acetylated form, generating eight different acetylated Tau (aTau) analogues. Similarly, phosphorylation sites found in normal brains and in Alzheimer's lesioned brains were considered to design phosphorylated Tau (pTau) analogues. All these designed variants were evaluated in intracellular fluid and near a microtubule (MT) model. Our in silico findings demonstrated that the electrostatic changes, due to the absence of positive Lys' charges in acetylation cases, or the increasingly negative charge in the phosphorylated forms, hamper the association to the MT tubulins in most cases. Post-translational modifications also pose very distinct conformations to the ones described for native Tau, which hinders the microtubule-binding region (MTBR) and turns difficult the expected binding. Our study elucidates important molecular processes behind Tau abnormal function which can inspire novel therapeutics to address Alzheimer's disease.

Keywords:  Acetylation; Alzheimer’s disease; Intrinsically disordered protein; Molecular dynamics simulations; Phosphorylation; Tau

DOI:  https://doi.org/10.1016/j.bbagen.2022.130276
Biomacromolecules. 2022 Nov 15.

Sequence Context and Complex Hofmeister Salt Interactions Dictate Phase Separation Propensity of Resilin-like Polypeptides.

James Brandt Otis, Simon Sharpe.

Resilin is an elastic material found in insects with exceptional durability, resilience, and extensibility, making it a promising biomaterial for tissue engineering. The monomeric precursor, pro-resilin, undergoes thermo-responsive self-assembly through liquid-liquid phase separation (LLPS). Understanding the molecular details of this assembly process is critical to developing complex biomaterials. The present study investigates the interplay between the solvent, sequence syntax, structure, and dynamics in promoting LLPS of resilin-like-polypeptides (RLPs) derived from domains 1 and 3 of Drosophila melanogaster pro-resilin. NMR, UV-vis, and microscopy data demonstrate that while kosmotropic salts and low pH promote LLPS, the effects of chaotropic salts with increasing pH are more complex. Subtle variations between the repeating amino acid motifs of resilin domain 1 and domain 3 lead to significantly different salt and pH dependence of LLPS, with domain 3 sequence motifs more strongly favoring phase separation under most conditions. These findings provide new insight into the molecular drivers of RLP phase separation and the complex roles of both RLP sequence and solution composition in fine-tuning assembly conditions.

DOI: https://doi.org/10.1021/acs.biomac.2c01027
Int J Biol Macromol. 2022 Nov 11. pii: S0141-8130(22)02618-6. [Epub ahead of print]

Binding of two zinc ions promotes liquid-liquid phase separation of Tau.

Dahbia Yatoui, Philipp O Tsvetkov, Romain La Rocca, Viktoriia E Baksheeva, Diane Allegro, Gilles Breuzard, Géraldine Ferracci, Deborah Byrne, François Devred.

  Tau is a naturally disordered microtubule associated protein which forms intraneuronal aggregates in several neurodegenerative diseases including Alzheimer's disease (AD). It was reported that zinc interaction with tau protein can trigger its aggregation. Recently we identified three zinc binding sites located in the N-terminal part, repeat region and the C-terminal part of tau. Here we characterized zinc binding to each of the three sites using isothermal titration calorimetry (ITC) and determined the impact of each site on aggregation using dynamic light scattering (DLS) assays. First, we confirmed the presence of three zinc binding sites on tau and determined the thermodynamic parameters of binding of zinc to these sites. We found a high-affinity zinc binding site located in the repeat region of tau and two N- and C-terminus binding sites with a lower binding constant for zinc. Second, we showed that tau aggregation necessitates zinc binding to the high affinity site in the R2R3 region, while LLPS necessitates zinc binding to any two binding sites. With regard to the role of zinc ions in the aggregation of proteins in neurodegenerative diseases, these findings bring new insights to the understanding of the aggregation mechanism of tau protein induced by zinc.

Keywords:  Aggregation; Liquid-liquid phase separation; Tau protein; Zinc

DOI:  https://doi.org/10.1016/j.ijbiomac.2022.11.060
PLoS Comput Biol. 2022 Nov 17. 18(11): e1010713

Soft disorder modulates the assembly path of protein complexes.

Beatriz Seoane, Alessandra Carbone.

The relationship between interactions, flexibility and disorder in proteins has been explored from many angles over the years: folding upon binding, flexibility of the core relative to the periphery, entropy changes, etc. In this work, we provide statistical evidence for the involvement of highly mobile and disordered regions in complex assembly. We ordered the entire set of X-ray crystallographic structures in the Protein Data Bank into hierarchies of progressive interactions involving identical or very similar protein chains, yielding 40205 hierarchies of protein complexes with increasing numbers of partners. We then examine them as proxies for the assembly pathways. Using this database, we show that upon oligomerisation, the new interfaces tend to be observed at residues that were characterised as softly disordered (flexible, amorphous or missing residues) in the complexes preceding them in the hierarchy. We also rule out the possibility that this correlation is just a surface effect by restricting the analysis to residues on the surface of the complexes. Interestingly, we find that the location of soft disordered residues in the sequence changes as the number of partners increases. Our results show that there is a general mechanism for protein assembly that involves soft disorder and modulates the way protein complexes are assembled. This work highlights the difficulty of predicting the structure of large protein complexes from sequence and emphasises the importance of linking predictors of soft disorder to the next generation of predictors of complex structure. Finally, we investigate the relationship between the Alphafold2's confidence metric pLDDT for structure prediction in unbound versus bound structures, and soft disorder. We show a strong correlation between Alphafold2 low confidence residues and the union of all regions of soft disorder observed in the hierarchy. This paves the way for using the pLDDT metric as a proxy for predicting interfaces and assembly paths.

DOI: https://doi.org/10.1371/journal.pcbi.1010713