Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19.

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation1. Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases2, whereas mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B553. Although the role of kinases in mitotic entry is well established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited4. Inhibition of PP2A:B55 is achieved by the intrinsically disordered proteins ARPP195,6 and FAM122A7. Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the single-particle cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies, both intrinsically disordered proteins bind PP2A:B55, but do so in highly distinct manners, leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provide a molecular roadmap for the development of therapeutic interventions for PP2A:B55-related diseases.

Advanced Sampling Methods for Multiscale Simulation of Disordered Proteins and Dynamic Interactions.

Intrinsically disordered proteins (IDPs) are highly prevalent and play important roles in biology and human diseases. It is now also recognized that many IDPs remain dynamic even in specific complexes and functional assemblies. Computer simulations are essential for deriving a molecular description of the disordered protein ensembles and dynamic interactions for a mechanistic understanding of IDPs in biology, diseases, and therapeutics. Here, we provide an in-depth review of recent advances in the multi-scale simulation of disordered protein states, with a particular emphasis on the development and application of advanced sampling techniques for studying IDPs. These techniques are critical for adequate sampling of the manifold functionally relevant conformational spaces of IDPs. Together with dramatically improved protein force fields, these advanced simulation approaches have achieved substantial success and demonstrated significant promise towards the quantitative and predictive modeling of IDPs and their dynamic interactions. We will also discuss important challenges remaining in the atomistic simulation of larger systems and how various coarse-grained approaches may help to bridge the remaining gaps in the accessible time- and length-scales of IDP simulations.

DIPEND: An Open-Source Pipeline to Generate Ensembles of Disordered Segments Using Neighbor-Dependent Backbone Preferences.

Ensemble-based structural modeling of flexible protein segments such as intrinsically disordered regions is a complex task often solved by selection of conformers from an initial pool based on their conformity to experimental data. However, the properties of the conformational pool are crucial, as the sampling of the conformational space should be sufficient and, in the optimal case, relatively uniform. In other words, the ideal sampling is both efficient and exhaustive. To achieve this, specialized tools are usually necessary, which might not be maintained in the long term, available on all platforms or flexible enough to be tweaked to individual needs. Here, we present an open-source and extendable pipeline to generate initial protein structure pools for use with selection-based tools to obtain ensemble models of flexible protein segments. Our method is implemented in Python and uses ChimeraX, Scwrl4, Gromacs and neighbor-dependent backbone distributions compiled and published previously by the Dunbrack lab. All these tools and data are publicly available and maintained. Our basic premise is that by using residue-specific, neighbor-dependent Ramachandran distributions, we can enhance the efficient exploration of the relevant region of the conformational space. We have also provided a straightforward way to bias the sampling towards specific conformations for selected residues by combining different conformational distributions. This allows the consideration of a priori known conformational preferences such as in the case of preformed structural elements. The open-source and modular nature of the pipeline allows easy adaptation for specific problems. We tested the pipeline on an intrinsically disordered segment of the protein Cd3ϵ and also a single-alpha helical (SAH) region by generating conformational pools and selecting ensembles matching experimental data using the CoNSEnsX+ server.

When Order Meets Disorder: Modeling and Function of the Protein Interface in Fuzzy Complexes.

The degree of proteins structural organization ranges from highly structured, compact folding to intrinsic disorder, where each degree of self-organization corresponds to specific functions: well-organized structural motifs in enzymes offer a proper environment for precisely positioned functional groups to participate in catalytic reactions; at the other end of the self-organization spectrum, intrinsically disordered proteins act as binding hubs via the formation of multiple, transient and often non-specific interactions. This review focusses on cases where structurally organized proteins or domains associate with highly disordered protein chains, leading to the formation of interfaces with varying degrees of fuzziness. We present a review of the computational methods developed to provide us with information on such fuzzy interfaces, and how they integrate experimental information. The discussion focusses on two specific cases, microtubules and homologous recombination nucleoprotein filaments, where a network of intrinsically disordered tails exerts regulatory function in recruiting partner macromolecules, proteins or DNA and tuning the atomic level association. Notably, we show how computational approaches such as molecular dynamics simulations can bring new knowledge to help bridging the gap between experimental analysis, that mostly concerns ensemble properties, and the behavior of individual disordered protein chains that contribute to regulation functions.

Structural Protein Analysis of Driver Gene Mutations in Conjunctival Melanoma.

In recent years, there has been tremendous enthusiasm with respect to detailing the genetic basis of many neoplasms, including conjunctival melanoma (CM). We aim to analyze five proteins associated with CM, namely BRAF, NRAS, c-KIT, NF1, and PTEN. We evaluated each protein for its intrinsically disordered protein regions (IDPRs) and its protein-protein interactions (PPI) with the Predictor of Natural Disordered Protein Regions (PONDR®) and the Search Tool for the Retrieval of Interacting Genes (STRING®). Our PONDR® analysis found high levels of IDPRs in all five proteins with mutations linked to CM. The highest levels of IDPRs were in BRAF (45.95%), followed by PTEN (31.76%), NF1 (22.19%), c-KIT (21.82%), and NRAS (14.81%). Our STRING analysis found that each of these five proteins had more predicted interactions then expected (p-value < 1.0 × 10-16). Our analysis demonstrates that the mutations linked to CM likely affected IDPRs and possibly altered their highly complex PPIs. Quantifying IDPRs in BRAF, NRAS, c-KIT, NF1, and PTEN and understanding these protein regions are important processes as IDPRs can be possible drug targets for novel targeted therapies for treating CM.

The N-terminal domain of the prion protein is required and sufficient for liquid-liquid phase separation: A crucial role of the Aß-binding domain.

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has been described for several pathogenic proteins linked to neurodegenerative diseases and is discussed as an early step in the formation of protein aggregates with neurotoxic properties. In prion diseases, neurodegeneration and formation of infectious prions is caused by aberrant folding of the cellular prion protein (PrPC). PrPC is characterized by a large intrinsically disordered N-terminal domain and a structured C-terminal globular domain. A significant fraction of mature PrPC is proteolytically processed in vivo into an entirely unstructured fragment, designated N1, and the corresponding C-terminal fragment C1 harboring the globular domain. Notably, N1 contains a polybasic motif that serves as a binding site for neurotoxic Aß oligomers. PrP can undergo LLPS; however, nothing is known how phase separation of PrP is triggered on a molecular scale. Here, we show that the intrinsically disordered N1 domain is necessary and sufficient for LLPS of PrP. Similar to full-length PrP, the N1 fragment formed highly dynamic liquid-like droplets. Remarkably, a slightly shorter unstructured fragment, designated N2, which lacks the Aß-binding domain and is generated under stress conditions, failed to form liquid-like droplets and instead formed amorphous assemblies of irregular structures. Through a mutational analysis, we identified three positively charged lysines in the postoctarepeat region as essential drivers of condensate formation, presumably largely via cation-π interactions. These findings provide insights into the molecular basis of LLPS of the mammalian prion protein and reveal a crucial role of the Aß-binding domain in this process.

A phase-separated nuclear GBPL circuit controls immunity in plants.

Liquid-liquid phase separation (LLPS) has emerged as a central paradigm for understanding how membraneless organelles compartmentalize diverse cellular activities in eukaryotes1-3. Here we identify a superfamily of plant guanylate-binding protein (GBP)-like GTPases (GBPLs) that assemble LLPS-driven condensates within the nucleus to protect against infection and autoimmunity. In Arabidopsis thaliana, two members of this family-GBPL1 and GBPL3-undergo phase-transition behaviour to control transcriptional responses as part of an allosteric switch that is triggered by exposure to biotic stress. GBPL1, a pseudo-GTPase, sequesters catalytically active GBPL3 under basal conditions but is displaced by GBPL3 LLPS when it enters the nucleus following immune cues to drive the formation of unique membraneless organelles termed GBPL defence-activated condensates (GDACs) that we visualized by in situ cryo-electron tomography. Within these mesoscale GDAC structures, native GBPL3 directly bound defence-gene promoters and recruited specific transcriptional coactivators of the Mediator complex and RNA polymerase II machinery to massively reprogram host gene expression for disease resistance. Together, our study identifies a GBPL circuit that reinforces the biological importance of phase-separated condensates, in this case, as indispensable players in plant defence.

NMR unveils an N-terminal interaction interface on acetylated-α-synuclein monomers for recruitment to fibrils.

Amyloid fibril formation of α-synuclein (αS) is associated with multiple neurodegenerative diseases, including Parkinson's disease (PD). Growing evidence suggests that progression of PD is linked to cell-to-cell propagation of αS fibrils, which leads to seeding of endogenous intrinsically disordered monomer via templated elongation and secondary nucleation. A molecular understanding of the seeding mechanism and driving interactions is crucial to inhibit progression of amyloid formation. Here, using relaxation-based solution NMR experiments designed to probe large complexes, we probe weak interactions of intrinsically disordered acetylated-αS (Ac-αS) monomers with seeding-competent Ac-αS fibrils and seeding-incompetent off-pathway oligomers to identify Ac-αS monomer residues at the binding interface. Under conditions that favor fibril elongation, we determine that the first 11 N-terminal residues on the monomer form a common binding site for both fibrils and off-pathway oligomers. Additionally, the presence of off-pathway oligomers within a fibril seeding environment suppresses seeded amyloid formation, as observed through thioflavin-T fluorescence experiments. This highlights that off-pathway αS oligomers can act as an auto-inhibitor against αS fibril elongation. Based on these data taken together with previous results, we propose a model in which Ac-αS monomer recruitment to the fibril is driven by interactions between the intrinsically disordered monomer N terminus and the intrinsically disordered flanking regions (IDR) on the fibril surface. We suggest that this monomer recruitment may play a role in the elongation of amyloid fibrils and highlight the potential of the IDRs of the fibril as important therapeutic targets against seeded amyloid formation.

The 'Shape-Shifter' Peptide from the Disulphide Isomerase PmScsC Shows Context-Dependent Conformational Preferences.

Multiple crystal structures of the homo-trimeric protein disulphide isomerase PmScsC reveal that the peptide which links the trimerization stalk and catalytic domain can adopt helical, ß-strand and loop conformations. This region has been called a 'shape-shifter' peptide. Characterisation of this peptide using NMR experiments and MD simulations has shown that it is essentially disordered in solution. Analysis of the PmScsC crystal structures identifies the role of intermolecular contacts, within an assembly of protein molecules, in stabilising the different linker peptide conformations. These context-dependent conformational properties may be important functionally, allowing for the binding and disulphide shuffling of a variety of protein substrates to PmScsC. They also have a relevance for our understanding of protein aggregation and misfolding showing how intermolecular quaternary interactions can lead to ß-sheet formation by a sequence that in other contexts adopts a helical structure. This 'shape-shifting' peptide region within PmScsC is reminiscent of one-to-many molecular recognition features (MoRFs) found in intrinsically disordered proteins which are able to adopt different conformations when they fold upon binding to their protein partners.

Intrinsic disorder in protein domains contributes to both organism complexity and clade-specific functions.

Interestingly, some protein domains are intrinsically disordered (abbreviated as IDD), and the disorder degree of same domains may differ in different contexts. However, the evolutionary causes and biological significance of these phenomena are unclear. Here, we address these issues by genome-wide analyses of the evolutionary and functional features of IDDs in 1,870 species across the three superkingdoms. As the result, there is a significant positive correlation between the proportion of IDDs and organism complexity with some interesting exceptions. These phenomena may be due to the high disorder of clade-specific domains and the different disorder degrees of the domains shared in different clades. The functions of IDDs are clade-specific and the higher proportion of post-translational modification sites may contribute to their complex functions. Compared with metazoans, fungi have more IDDs with a consecutive disorder region but a low disorder ratio, which reflects their different functional requirements. As for disorder variation, it's greater for domains among different proteins than those within the same proteins. Some clade-specific 'no-variation' or 'high-variation' domains are involved in clade-specific functions. In sum, intrinsic domain disorder is related to both the organism complexity and clade-specific functions. These results deepen the understanding of the evolution and function of IDDs.

Redox-mediated regulation of low complexity domain self-association.

Eukaryotic cells express thousands of protein domains long believed to function in the absence of molecular order. These intrinsically disordered protein (IDP) domains are typified by gibberish-like repeats of only a limited number of amino acids that we refer to as domains of low sequence complexity. A decade ago, it was observed that these low complexity (LC) domains can undergo phase transition out of aqueous solution to form either liquid-like droplets or hydrogels. The self-associative interactions responsible for phase transition involve the formation of specific cross-ß structures that are unusual in being labile to dissociation. Here we give evidence that the LC domains of two RNA binding proteins, ataxin-2 and TDP43, form cross-ß interactions that specify biologically relevant redox sensors.

Micellar TIA1 with folded RNA binding domains as a model for reversible stress granule formation.

TIA1, a protein critical for eukaryotic stress response and stress granule formation, is structurally characterized in full-length form. TIA1 contains three RNA recognition motifs (RRMs) and a C-terminal low-complexity domain, sometimes referred to as a "prion-related domain" or associated with amyloid formation. Under mild conditions, full-length (fl) mouse TIA1 spontaneously oligomerizes to form a metastable colloid-like suspension. RRM2 and RRM3, known to be critical for function, are folded similarly in excised domains and this oligomeric form of apo fl TIA1, based on NMR chemical shifts. By contrast, the termini were not detected by NMR and are unlikely to be amyloid-like. We were able to assign the NMR shifts with the aid of previously assigned solution-state shifts for the RRM2,3 isolated domains and homology modeling. We present a micellar model of fl TIA1 wherein RRM2 and RRM3 are colocalized, ordered, hydrated, and available for nucleotide binding. At the same time, the termini are disordered and phase separated, reminiscent of stress granule substructure or nanoscale liquid droplets.

Physicochemical Characterisation of KEIF-The Intrinsically Disordered N-Terminal Region of Magnesium Transporter A.

Magnesium transporter A (MgtA) is an active transporter responsible for importing magnesium ions into the cytoplasm of prokaryotic cells. This study focuses on the peptide corresponding to the intrinsically disordered N-terminal region of MgtA, referred to as KEIF. Primary-structure and bioinformatic analyses were performed, followed by studies of the undisturbed single chain using a combination of techniques including small-angle X-ray scattering, circular dichroism spectroscopy, and atomistic molecular-dynamics simulations. Moreover, interactions with large unilamellar vesicles were investigated by using dynamic light scattering, laser Doppler velocimetry, cryogenic transmission electron microscopy, and circular dichroism spectroscopy. KEIF was confirmed to be intrinsically disordered in aqueous solution, although extended and containing little ß -structure and possibly PPII structure. An increase of helical content was observed in organic solvent, and a similar effect was also seen in aqueous solution containing anionic vesicles. Interactions of cationic KEIF with anionic vesicles led to the hypothesis that KEIF adsorbs to the vesicle surface through electrostatic and entropic driving forces. Considering this, there is a possibility that the biological role of KEIF is to anchor MgtA in the cell membrane, although further investigation is needed to confirm this hypothesis.

Prediction of the Disordered Regions of Intrinsically Disordered Proteins Based on the Molecular Functions.

BACKGROUND: Intrinsically disordered proteins lack a well-defined three dimensional structure under physiological conditions while possessing the essential biological functions. They take part in various physiological processes such as signal transduction, transcription and posttranslational modifications and etc. The disordered regions are the main functional sites for intrinsically disordered proteins. Therefore, the research of the disordered regions has become a hot issue. OBJECTIVE: In this paper, our motivation is to analysis of the features of disordered regions with different molecular functions and predict of different disordered regions using valid features. METHODS: In this article, according to the different molecular function, we firstly divided intrinsically disordered proteins into six classes in DisProt database. Then, we extracted four features using bioinformatics methods, namely, Amino Acid Index (AAIndex), codon frequency (Codon), three kinds of protein secondary structure compositions (3PSS) and Chemical Shifts (CSs), and used these features to predict the disordered regions of the different functions by Support Vector Machine (SVM). RESULTS: The best overall accuracy was 99.29% using the chemical shift (CSs) as feature. In feature fusion, the overall accuracy can reach 88.70% by using CSs+AAIndex as features. The overall accuracy was up to 86.09% by using CSs+AAIndex+Codon+3PSS as features. CONCLUSION: We predicted and analyzed the disordered regions based on the molecular functions. The results showed that the prediction performance can be improved by adding chemical shifts and AAIndex as features, especially chemical shifts. Moreover, the chemical shift was the most effective feature in the prediction. We hoped that our results will be constructive for the study of intrinsically disordered proteins.

DEPICTER: Intrinsic Disorder and Disorder Function Prediction Server.

Computational predictions of the intrinsic disorder and its functions are instrumental to facilitate annotation for the millions of unannotated proteins. However, access to these predictors is fragmented and requires substantial effort to find them and to collect and combine their results. The DEPICTER (DisorderEd PredictIon CenTER) server provides first-of-its-kind centralized access to 10 popular disorder and disorder function predictions that cover protein and nucleic acids binding, linkers, and moonlighting regions. It automates the prediction process, runs user-selected methods on the server side, visualizes the results, and outputs all predictions in a consistent and easy-to-parse format. DEPICTER also includes two accurate consensus predictors of disorder and disordered protein binding. Empirical tests on an independent (low similarity) benchmark dataset reveal that the computational tools included in DEPICTER generate accurate predictions that are significantly better than the results secured using sequence alignment. The DEPICTER server is freely available at http://biomine.cs.vcu.edu/servers/DEPICTER/.

Do sequence neighbours of intrinsically disordered regions promote structural flexibility in intrinsically disordered proteins?

Intrinsically disordered proteins (IDPs) are crucial players in various cellular activities. Several experimental and computational analyses have been conducted to study structural pliability and functional potential of IDPs. In spite of active research in past few decades, what induces structural disorder in IDPs and how is still elusive. Many studies testify that sequential and spatial neighbours often play important roles in determining structural and functional behaviour of proteins. Considering this fact, we assessed sequence neighbours of intrinsically disordered regions (IDRs) to understand if they have any role to play in inducing structural flexibility in IDPs. Our analysis includes 97% eukaryotic IDPs and 3% from bacteria and viruses. Physicochemical and structural parameters including amino acid propensity, hydrophobicity, secondary structure propensity, relative solvent accessibility, B-factor and atomic packing density are used to characterise the neighbouring residues of IDRs (NRIs). We show that NRIs exhibit a unique nature, which makes them stand out from both ordered and disordered residues. They show correlative occurrences of residue pairs like Ser-Thr and Gln-Asn, indicating their tendency to avoid strong biases of order or disorder promoting amino acids. We also find differential preferences of amino acids between N- and C-terminal neighbours, which might indicate a plausible directional effect on the dynamics of adjacent IDRs. We designed an efficient prediction tool using Random Forest to distinguish the NRIs from the ordered residues. Our findings will contribute to understand the behaviour of IDPs, and may provide potential lead in deciphering the role of IDRs in protein folding and assembly.

The Functionally Important N-Terminal Half of Fission Yeast Mid1p Anillin Is Intrinsically Disordered and Undergoes Phase Separation.

Division of fungal and animal cells depends on scaffold proteins called anillins. Cytokinesis by the fission yeast Schizosaccharomyces pombe is compromised by the loss of anillin Mid1p (Mid1, UniProtKB P78953 ), because cytokinesis organizing centers, called nodes, are misplaced and fail to acquire myosin-II, so they assemble slowly into abnormal contractile rings. The C-terminal half of Mid1p consists of lipid binding C2 and PH domains, but the N-terminal half (Mid1p-N452) performs most of the functions of the full-length protein. Little is known about the structure of the N-terminal half of Mid1p, so we investigated its physical properties using structure prediction tools, spectroscopic techniques, and hydrodynamic measurements. The data indicate that Mid1p-N452 is intrinsically disordered but moderately compact. Recombinant Mid1p-N452 purified from insect cells was phosphorylated, which weakens its tendency to aggregate. Purified Mid1p-N452 demixes into liquid droplets at concentrations far below its concentration in nodes. These physical properties are appropriate for scaffolding other proteins in nodes.

A curvature-dependent membrane binding by tyrosine kinase Fer involves an intrinsically disordered region.

Tyrosine kinases are important enzymes that mediate signal transduction at the plasma membrane. While the significance of membrane localization of tyrosine kinases has been well evaluated, the role of membrane curvature in their regulation is unknown. Here, we demonstrate that an intrinsically disordered region in the tyrosine kinase Fer acts as a membrane curvature sensor that preferentially binds to highly curved membranes in vitro. This region forms an amphipathic α-helix upon interaction with curved membranes, aligning hydrophobic residues on one side of the helical structure. Further, the tyrosine kinase activity of Fer is significantly enhanced by the membrane in a manner dependent on curvature. We propose a model for the regulation of Fer based on an intramolecular interaction and the curvature-dependent membrane binding mediated by its intrinsically disordered region.

Structural Characterization of Highly Flexible Proteins by Small-Angle Scattering.

Intrinsically Disordered Proteins (IDPs) are fundamental actors of biological processes. Their inherent plasticity facilitates very specialized tasks in cell regulation and signalling, and their malfunction is linked to severe pathologies. Understanding the functional role of disorder requires the structural characterization of IDPs and the complexes they form. Small-angle Scattering of X-rays (SAXS) and Neutrons (SANS) have notably contributed to this structural understanding. In this review we summarize the most relevant developments in the field of SAS studies of disordered proteins. Emphasis is given to ensemble methods and how SAS data can be combined with computational approaches or other biophysical information such as NMR. The unique capabilities of SAS enable its application to extremely challenging disordered systems such as low-complexity regions, amyloidogenic proteins and transient biomolecular complexes. This reinforces the fundamental role of SAS in the structural and dynamic characterization of this elusive family of proteins.

From Disorder to Mis-Order: Structural Aspects of Pathogenic Oligomerization in Conformational Diseases.

Proteins implicated in neurological conformational diseases contain substantial amounts of "intrinsic disorder". These native monomeric functional states may transit into some oligomeric states that have high ß-sheet contents and seed the formation of insoluble amyloid fibrils. The prevailing view is that these "toxic" oligomers should be targeted for drug development. Here, an overview of the diseases was presented, within the general framework of the oligomerization of intrinsically disordered proteins. These systems pose some specific challenges to structural studies: the toxic oligomers are transient, low in concentration, and often need to be studied in a heterogeneous environment. Nevertheless, there have been much exciting progress as a result of the creative use of experimental techniques, a selection of these were outlined.