Intrinsically Disordered Proteins Research Articles

Wild-Type α-Synuclein Structure and Aggregation: A Comprehensive Coarse-Grained and All-Atom Molecular Dynamics Study.

α-Synuclein (α-syn) is a 140 amino acid intrinsically disordered protein (IDP) and the primary component of cytotoxic oligomers implicated in the etiology of Parkinson's disease (PD). While IDPs lack a stable three-dimensional structure, they sample a heterogeneous ensemble of conformations that can, in principle, be assessed through molecular dynamics simulations. However, describing the structure and aggregation of large IDPs is challenging due to force field (FF) accuracy and sampling limitations. To cope with the latter, coarse-grained (CG) FFs emerge as a potential alternative at the expense of atomic detail loss. Whereas CG models can accurately describe the structure of the monomer, less is known about aggregation. The latter is key for assessing aggregation pathways and designing aggregation inhibitor drugs. Herein, we investigate the structure and dynamics of α-syn using different resolution CG (Martini3 and Sirah2) and all-atom (Amber99sb and Charmm36m) FFs to gain insight into the differences and resemblances between these models. The dependence of the magnitude of protein-water interactions and the putative need for enhanced sampling (replica exchange) methods in CG simulations are analyzed to distinguish between force field accuracy and sampling limitations. The stability of the CG models of an α-syn fibril was also investigated. Additionally, α-syn aggregation was studied through umbrella sampling for the CG models and CG/all-atom models for an 11-mer peptide (NACore) from an amyloidogenic domain of α-syn. Our results show that despite the α-syn structures of Martini3 and Sirah2 with enhanced protein-water interactions being similar, major differences exist concerning aggregation. The Martini3 fibril is not stable, and the binding free energy of α-syn and NACore is positive, opposite to Sirah2. Sirah2 peptides in a zwitterionic form, in turn, display termini interactions that are too strong, resulting in end-to-end orientation. Sirah2, with enhanced protein-water interactions and neutral termini, provides, however, a peptide aggregation free energy profile similar to that found with all-atom models. Overall, we find that Sirah2 with enhanced protein-water interactions is suitable for studying protein-protein and protein-drug aggregation.

Blending and separating dynamics of RNA-binding proteins develop architectural splicing networks spreading throughout the nucleus

The eukaryotic nucleus has a highly organized structure. Although the spatiotemporal arrangement of spliceosomes on nascent RNA drives splicing, the nuclear architecture that directly supports this process remains unclear. Here, we show that RNA-binding proteins (RBPs) assembled on RNA form meshworks in human and mouse cells. Core and accessory RBPs in RNA splicing make two distinct meshworks adjacently but distinctly distributed throughout the nucleus. This is achieved by mutual exclusion dynamics between the charged and uncharged intrinsically disordered regions (IDRs) of RBPs. These two types of meshworks compete for spatial occupancy on pre-mRNA to regulate splicing. Furthermore, the optogenetic enhancement of the RBP meshwork causes aberrant splicing, particularly of genes involved in neurodegeneration. Genetic mutations associated with neurodegenerative diseases are often found in the IDRs of RBPs, and cells harboring these mutations exhibit impaired meshwork formation. Our results uncovered the spatial organization of RBP networks to drive RNA splicing.

Computational insights into intrinsically disordered regions in protein-nucleic acid complexes

We study transitions in intrinsically disordered regions (IDRs) upon complex formation, utilizing X-ray-solved structural dataset of protein-DNA and protein-RNA complexes, along with their available unbound protein forms. The identified IDRs are categorized into three classes: Disordered-to-Ordered (D-O), Disordered-to-Partial Ordered (D-PO) and Disordered-to-Disordered (D-D) after comparing them in unbound and complex forms. In the D-O class, IDRs form secondary structures like coils, helices, and strands upon binding to nucleic acids. Though a majority of these IDRs are present at the surface of the complexes, a significant number of IDRs are also observed at the interfaces and are involved in polar interactions. The hydrogen bonds made by the interface IDRs (B_IDRs) with phosphates and bases of nucleic acids are comparatively more than those formed with sugars. B_IDRs form more H-bonds with the ribose in protein-RNA than with the deoxyribose in protein-DNA. Among the B_IDRs, Arg and Lys prefer to interact with the major and minor grooves of DNA and RNA, respectively. Ser, however, prefers the minor groove in both the nucleic acids. Interestingly, we report 61 and 48 IDRs in 31 protein-DNA and 22 protein-RNA complexes, respectively, suggesting nucleic acid binding to proteins may also result in ordered-to-disordered transitions.

Diffusing protein binders to intrinsically disordered proteins.

Proteins which bind intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) with high affinity and specificity could have considerable utility for therapeutic and diagnostic applications. However, a general methodology for targeting IDPs/IDRs has yet to be developed. Here, we show that starting only from the target sequence of the input, and freely sampling both target and binding protein conformation, RFdiffusion can generate binders to IDPs and IDRs in a wide range of conformations. We use this approach to generate binders to the IDPs Amylin, C-peptide and VP48 in a range of conformations with Kds in the 3 -100nM range. The Amylin binder inhibits amyloid fibril formation and dissociates existing fibers, and enables enrichment of amylin for mass spectrometry-based detection. For the IDRs G3bp1, common gamma chain (IL2RG) and prion, we diffused binders to beta strand conformations of the targets, obtaining 10 to 100 nM affinity. The IL2RG binder colocalizes with the receptor in cells, enabling new approaches to modulating IL2 signaling. Our approach should be widely useful for creating binders to flexible IDPs/IDRs spanning a wide range of intrinsic conformational preferences.

Sequence-specific targeting of intrinsically disordered protein regions.

A general approach to design proteins that bind tightly and specifically to intrinsically disordered regions (IDRs) of proteins and flexible peptides would have wide application in biological research, therapeutics, and diagnosis. However, the lack of defined structures and the high variability in sequence and conformational preferences has complicated such efforts. We sought to develop a method combining biophysical principles with deep learning to readily generate binders for any disordered sequence. Instead of assuming a fixed regular structure for the target, general recognition is achieved by threading the query sequence through diverse extended binding modes in hundreds of templates with varying pocket depths and spacings, followed by RFdiffusion refinement to optimize the binder-target fit. We tested the method by designing binders to 39 highly diverse unstructured targets. Experimental testing of ∼36 designs per target yielded binders with affinities better than 100 nM in 34 cases, and in the pM range in four cases. The co-crystal structure of a designed binder in complex with dynorphin A is closely consistent with the design model. All by all binding experiments for 20 designs binding diverse targets show they are highly specific for the intended targets, with no crosstalk even for the closely related dynorphin A and dynorphin B. Our approach thus could provide a general solution to the intrinsically disordered protein and peptide recognition problem.

Evolutional heterochromatin condensation delineates chromocenter formation and retrotransposon silencing in plants.

Heterochromatic condensates (chromocenters) are critical for maintaining the silencing of heterochromatin. It is therefore puzzling that the presence of chromocenters is variable across plant species. Here we reveal that variations in the plant heterochromatin protein ADCP1 confer a diversity in chromocenter formation via phase separation. ADCP1 physically interacts with the high mobility group protein HMGA to form a complex and mediates heterochromatin condensation by multivalent interactions. The loss of intrinsically disordered regions (IDRs) in ADCP1 homologues during evolution has led to the absence of prominent chromocenter formation in various plant species, and introduction of IDR-containing ADCP1 with HMGA promotes heterochromatin condensation and retrotransposon silencing. Moreover, plants in the Cucurbitaceae group have evolved an IDR-containing chimaera of ADCP1 and HMGA, which remarkably enables formation of chromocenters. Together, our work uncovers a coevolved mechanism of phase separation in packing heterochromatin and silencing retrotransposons.

Proteome-scale characterisation of motif-based interactome rewiring by disease mutations

Whole genome and exome sequencing are reporting on hundreds of thousands of missense mutations. Taking a pan-disease approach, we explored how mutations in intrinsically disordered regions (IDRs) break or generate protein interactions mediated by short linear motifs. We created a peptide-phage display library tiling ~57,000 peptides from the IDRs of the human proteome overlapping 12,301 single nucleotide variants associated with diverse phenotypes including cancer, metabolic diseases and neurological diseases. By screening 80 human proteins, we identified 366 mutation-modulated interactions, with half of the mutations diminishing binding, and half enhancing binding or creating novel interaction interfaces. The effects of the mutations were confirmed by affinity measurements. In cellular assays, the effects of motif-disruptive mutations were validated, including loss of a nuclear localisation signal in the cell division control protein CDC45 by a mutation associated with Meier-Gorlin syndrome. The study provides insights into how disease-associated mutations may perturb and rewire the motif-based interactome.

An extended interaction site determines binding between AP180 and AP2 in clathrin mediated endocytosis

The early phases of clathrin mediated endocytosis are organized through a highly complex interaction network mediated by clathrin associated sorting proteins (CLASPs) that comprise long intrinsically disordered regions (IDRs). AP180 is a CLASP exclusively expressed in neurons and comprises a long IDR of around 600 residues, whose function remains partially elusive. Using NMR spectroscopy, we discovered an extended and strong interaction site within AP180 with the major adaptor protein AP2, and describe its binding dynamics at atomic resolution. We find that the 70 residue-long site determines the overall interaction between AP180 and AP2 in a dynamic equilibrium between its bound and unbound states, while weaker binding sites contribute to the overall affinity at much higher concentrations of AP2. Our data suggest that this particular interaction site might play a central role in recruitment of adaptors to the clathrin coated pit, whereas more transient and promiscuous interactions allow reshaping of the interaction network until cargo uptake inside a coated vesicle.

Transcription Factor Condensates Mediate Clustering of MET Regulon and Enhancement in Gene Expression.

Some transcription factors (TFs) can form liquid-liquid phase separated (LLPS) condensates. However, the functions of these TF condensates in 3D genome organization and gene regulation remain elusive. In response to methionine (met) starvation, budding yeast TF Met4 and a few co-activators, including Met32, induce a set of genes involved in met biosynthesis. Here, we show that the endogenous Met4 and Met32 form co-localized puncta-like structures in yeast nuclei upon met depletion. Recombinant Met4 and Met32 form mixed droplets with LLPS properties in vitro. In relation to chromatin, Met4 puncta co-localize with target genes, and at least a subset of these target genes is clustered in 3D in a Met4-dependent manner. A MET3pr-GFP reporter inserted near several native Met4 binding sites becomes co-localized with Met4 puncta and displays enhanced transcriptional activity. A Met4 variant with a partial truncation of an intrinsically disordered region (IDR) shows less puncta formation, and this mutant selectively reduces the reporter activity near Met4 binding sites to the basal level. Overall, these results support a model where Met4 and co-activators form condensates to bring multiple target genes into a vicinity with higher local TF concentrations, which facilitates a strong response to methionine depletion.

Biomolecular condensates can function as inherent catalysts.

We report the discovery that chemical reactions such as ATP hydrolysis can be catalyzed by condensates formed by intrinsically disordered proteins (IDPs), which themselves lack any intrinsic ability to function as enzymes. This inherent catalytic feature of condensates derives from the electrochemical environments and the electric fields at interfaces that are direct consequences of phase separation. The condensates we studied were capable of catalyzing diverse hydrolysis reactions, including hydrolysis and radical-dependent breakdown of ATP whereby ATP fully decomposes to adenine and multiple carbohydrates. This distinguishes condensates from naturally occurring ATPases, which can only catalyze the dephosphorylation of ATP. Interphase and interfacial properties of condensates can be tuned via sequence design, thus enabling control over catalysis through sequence-dependent electrochemical features of condensates. Incorporation of hydrolase-like synthetic condensates into live cells enables activation of transcriptional circuits that depend on products of hydrolysis reactions. Inherent catalytic functions of condensates, which are emergent consequences of phase separation, are likely to affect metabolic regulation in cells.

O-glycosylation of intrinsically disordered regions regulates homeostasis of membrane proteins in streptococci.

Proteins harboring intrinsically disordered regions (IDRs) lacking stable secondary or tertiary structures are abundant across the three domains of life. These regions have not been systematically studied in prokaryotes. Our genome-wide analysis identifies extracytoplasmic serine/threonine-rich IDRs in several biologically important membrane proteins in streptococci. We demonstrate that these IDRs are O-glycosylated with glucose by glycosyltransferases GtrB and PgtC2 in Streptococcus pyogenes and Streptococcus pneumoniae, and with N-acetylgalactosamine by a Pgf-dependent mechanism in Streptococcus mutans. Absence of glycosylation leads to a defect in biofilm formation under ethanol-stressed conditions in S. mutans. We link this phenotype to the C-terminal IDR of a post-translocation secretion chaperone PrsA. O-glycosylation of the IDR protects this region from proteolytic degradation. The IDR length attenuates the efficiency of glycosylation and, consequently, the expression level of PrsA. Taken together, our data reveal that O-glycosylation of IDRs functions as a dynamic switch of protein homeostasis in streptococci.

IDRWalker: A Random Walk Based Tool for Generating Intrinsically Disordered Regions in Large Protein Complexes.

Intrinsically disordered regions (IDRs), which may be functionally important, are common in proteins. However, the structures of IDRs are often missing due to their highly dynamic nature. In the study of IDRs, integrative modeling combining computational simulations and experimental data is a common approach, for which initial structures of the IDRs need to be built. However, applying this method to large protein complexes is challenging because existing structure generation tools are sometimes unsuitable for IDRs in large systems. To facilitate convenient and rapid structure generation of IDRs in large protein complexes, we developed a computational tool named IDRWalker based on self-avoiding random walks. Three protein complexes were used to illustrate the efficiency of the tool, and it was found that IDRs in more than 800 chains of the nuclear pore complex could be generated in minutes. These structures of large protein complexes with added IDRs can be further used to run computational simulations for integrative modeling.

Coarse-Grained Modeling of Liquid-Liquid Phase Separation in Cells: Challenges and Opportunities.

Liquid-liquid phase separation (LLPS) within cells gives rise to membraneless organelles, which play pivotal roles in numerous cellular functions. A comprehensive understanding of the functional aspects of intrinsically disordered protein (IDP) condensates necessitates elucidating their inherent structures and establishing correlations with biological functions. Coarse-grained (CG) molecular dynamics (MD) simulations present a promising avenue for gaining insights into LLPS mechanisms of biomacromolecules. Essential to this endeavor is the development of tailored CG force fields for MD simulations, incorporating the full spectrum of biomolecules involved in the formation of condensates and accounting for real-time biochemical reactions coupled to the LLPS. Moreover, developing accurate theoretical frameworks and establishing links between condensate structure and its function are imperative for a thorough comprehension of LLPS of biological systems.

Structural basis of the human transcriptional Mediator complex modulated by its dissociable Kinase module.

The eukaryotic Mediator, comprising a large Core (cMED) and a dissociable CDK8 kinase module (CKM), regulates RNA Polymerase II (Pol II)-dependent transcription. cMED recruits Pol II and promotes pre-initiation complex (PIC) formation in a manner inhibited by the CKM, which is also implicated in post-initiation control of gene expression. Herein we report cryo-electron microscopy structures of the human complete Mediator and its CKM, which explains the basis for CKM inhibition of cMED-activated transcription. The CKM binds to cMED through an intrinsically disordered region (IDR) in MED13 and HEAT repeats in MED12. The CKM inhibits transcription by allocating its MED13 IDR to occlude binding of Pol II and MED26 to cMED and further obstructing cMED-PIC assembly through steric hindrance with TFIIH and the +1 nucleosome. Notably, MED12 binds to the cMED Hook, positioning CDK8 downstream of the transcription start site, which sheds new light on its stimulatory function in post-initiation events.

Dimerization of Full‐length Aβ ‐42 Peptide: A Comparison of Different Force Fields and WaterModels

Among the two isoforms of amyloid‐ i.e., Aβ‐40 and Aβ‐42, Aβ‐42 is more toxic due to its increased aggregation propensity. The oligomerization pathways of amyloid‐β may be investigated by studying its dimerization process at an atomic level. Intrinsically disordered proteins (IDPs) lack well‐defined structures and are associated with numerous neurodegenerative disorders. Molecular dynamics simulations of these proteins are often limited by the choice of parameters due to inconsistencies in the empirically developed protein force fields and water models. To evaluate the accuracy of recently developed force fields for IDPs, we study the dimerization of full‐length Aβ‐42 in aqueous solution with three different combinations of AMBER force field parameters and water models such as ff14SB/TIP3P, ff19SB/OPC, and ff19SB/TIP3P using classical MD and Umbrella Sampling method. This work may be used as a benchmark to compare the performance of different force fields for the simulations of IDPs.

An IDR-dependent mechanism for nuclear receptor control of Mediator interaction with RNA polymerase II

The essential Mediator (MED) coactivator complex plays a well-understood role in regulation of basal transcription in all eukaryotes, but the mechanism underlying its role in activator-dependent transcription remains unknown. We investigated modulation of metazoan MED interaction with RNA polymerase II (RNA Pol II) by antagonistic effects of the MED26 subunit and the CDK8 kinase module (CKM). Biochemical analysis of CKM-MED showed that the CKM blocks binding of the RNA Pol II carboxy-terminal domain (CTD), preventing RNA Pol II interaction. This restriction is eliminated by nuclear receptor (NR) binding to CKM-MED, which enables CTD binding in a MED26-dependent manner. Cryoelectron microscopy (cryo-EM) and crosslinking-mass spectrometry (XL-MS) revealed that the structural basis for modulation of CTD interaction with MED relates to a large intrinsically disordered region (IDR) in CKM subunit MED13 that blocks MED26 and CTD interaction with MED but is repositioned upon NR binding. Hence, NRs can control transcription initiation by priming CKM-MED for MED26-dependent RNA Pol II interaction.

A curated rotamer library for common post-translational modifications of proteins.

Sidechain rotamer libraries of the common amino acids of a protein are useful for folded protein structure determination and for generating ensembles of intrinsically disordered proteins (IDPs). However, much of protein function is modulated beyond the translated sequence through the introduction of post-translational modifications (PTMs). In this work, we have provided a curated set of side chain rotamers for the most common PTMs derived from the RCSB PDB database, including phosphorylated, methylated, and acetylated sidechains. Our rotamer libraries improve upon existing methods such as SIDEpro, Rosetta, and AlphaFold3 in predicting the experimental structures for PTMs in folded proteins. In addition, we showcase our PTM libraries in full use by generating ensembles with the Monte Carlo Side Chain Entropy (MCSCE) for folded proteins, and combining MCSCE with the Local Disordered Region Sampling algorithms within IDPConformerGenerator for proteins with intrinsically disordered regions. The codes for dihedral angle computations and library creation are available at https://github.com/THGLab/ptm_sc.git.

Sampling Conformational Ensembles of Highly Dynamic Proteins via Generative Deep Learning.

Proteins are inherently dynamic, and their conformational ensembles are functionally important in biology. Large-scale motions may govern protein structure-function relationship, and numerous transient but stable conformations of intrinsically disordered proteins (IDPs) can play a crucial role in biological function. Investigating conformational ensembles to understand regulations and disease-related aggregations of IDPs is challenging both experimentally and computationally. In this paper we first introduced an unsupervised deep learning-based model, termed Internal Coordinate Net (ICoN), which learns the physical principles of conformational changes from molecular dynamics (MD) simulation data. Second, we selected interpolating data points in the learned latent space that rapidly identify novel synthetic conformations with sophisticated and large-scale sidechains and backbone arrangements. Third, with the highly dynamic amyloid-β 1-42 (Aβ42) monomer, our deep learning model provided a comprehensive sampling of Aβ42's conformational landscape. Analysis of these synthetic conformations revealed conformational clusters that can be used to rationalize experimental findings. Additionally, the method can identify novel conformations with important interactions in atomistic details that are not included in the training data. New synthetic conformations showed distinct sidechain rearrangements that are probed by our EPR and amino acid substitution studies. This approach is highly transferable and can be used for any available data for training. The work also demonstrated the ability for deep learning to utilize learned natural atomistic motions in protein conformation sampling.

Modulation of biophysical properties of nucleocapsid protein in the mutant spectrum of SARS-CoV-2.

Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also observe functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.

Modulation of biophysical properties of nucleocapsid protein in the mutant spectrum of SARS-CoV-2

Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also observe functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.