Intrinsically Disordered Proteins Research Articles

Intrinsic stiffness and Θ-solvent regime in intrinsically disordered proteins: Implications for liquid-liquid phase separation

Abstract Liquid-liquid phase separation (LLPS) exhibited by intrinsically disordered proteins (IDPs) depends on the solvation state around the Θ-regime, which separates good from poor solvent. Experimentally, the Θ-solvent regime of the finite length (N) IDPs, as probed by small angle X-ray scattering (SAXS) and single molecular fluorescence resonance energy transfer (smFRET), is in disagreement. Using computer simulations of a coarse-grained IDP model, we address the effect of chain length on the Θ-regime of IDPs with polar side chains (polyglutamine) and hydrophobic side chains (polyleucine) subject to varying concentrations of cosolvents [C], urea (denaturant) or trimethylamine N-oxide (protective osmolyte) in water. Due to their intrinsic stiffness, these IDPs are always expanded on short-length scales, independent of the solvent quality. As a result, for short IDP sequences (≈ 10 to 25 residues), their propensity to exhibit LLPS cannot be inferred from single-chain properties. Further, for finite-size IDPs, the cosolvent concentration to attain the Θ-regime ([CΘ]) extracted from the structure factor emulating SAXS and pair distances mimicking smFRET differs. They converge to the same cosolvent concentration only at large N, indicating that finite size corrections vary for different IDP properties. We show that the radius of gyration (Rg) of the IDPs in the Θ-solvent regime satisfies the scaling relation Rg2 = N f(cN), which can be exploited to accurately extract [CΘ] (c = ([C]/[CΘ]-1)). We demonstrate the importance of finite size aspects originating from the chain stiffness and thermal blob size in analyzing IDP properties to identify the Θ-solvent regime.

Epigene functional diversity: isoform usage, disordered domain content, and variable binding partners

BackgroundEpigenes are defined as proteins that perform post-translational modification of histones or DNA, reading of post-translational modifications, form complexes with epigenetic factors or changing the general structure of chromatin. This specialized group of proteins is responsible for controlling the organization of genomic DNA in a cell-type specific fashion, controlling normal development in a spatial and temporal fashion. Moreover, mutations in epigenes have been implicated as causal in germline pediatric disorders and as driver mutations in cancer. Despite their importance to human disease, to date, there has not been a systematic analysis of the sources of functional diversity for epigenes at large. Epigenes’ unique functions that require the assembly of pools within the nucleus suggest that their structure and amino acid composition would have been enriched for features that enable efficient assembly of chromatin and DNA for transcription, splicing, and post-translational modifications.ResultsIn this study, we assess the functional diversity stemming from gene structure, isoforms, protein domains, and multiprotein complex formation that drive the functions of established epigenes. We found that there are specific structural features that enable epigenes to perform their variable roles depending on the cellular and environmental context. First, epigenes are significantly larger and have more exons compared with non-epigenes which contributes to increased isoform diversity. Second epigenes participate in more multimeric complexes than non-epigenes. Thirdly, given their proposed importance in membraneless organelles, we show epigenes are enriched for substantially larger intrinsically disordered regions (IDRs). Additionally, we assessed the specificity of their expression profiles and showed epigenes are more ubiquitously expressed consistent with their enrichment in pediatric syndromes with intellectual disability, multiorgan dysfunction, and developmental delay. Finally, in the L1000 dataset, we identify drugs that can potentially be used to modulate expression of these genes.ConclusionsHere we identify significant differences in isoform usage, disordered domain content, and variable binding partners between human epigenes and non-epigenes using various functional genomics datasets from Ensembl, ENCODE, GTEx, HPO, LINCS L1000, and BrainSpan. Our results contribute new knowledge to the growing field focused on developing targeted therapies for diseases caused by epigene mutations, such as chromatinopathies and cancers.

Identifying Subtypes of Cannabis Use Disorder and Cannabis Consumption Predictors among Daily Cannabis Consumers

Identifying Subtypes of Cannabis Use Disorder and Cannabis Consumption Predictors among Daily Cannabis Consumers

ATP Binding and Inhibition of Intrinsically Disordered Protein Interactions.

Recent studies have shown that ATP at high physiological concentrations (>5 mM) can inhibit liquid-liquid phase separation (LLPS) driven by interactions between intrinsically disordered proteins (IDPs). However, the mechanism underlying such inhibitory effect still remains elusive. Here, we used all-atom molecular dynamics simulation to study the interaction of ATP with two typical IDPs (i.e., FUS PLD and RGG domain of hnRNP G), and its impacts on IDP interactions. ATP exhibits a considerable tendency to bind to both IDPs and effectively inhibits their interactions. For the RGG domain, Arg residues are critical for both ATP binding and IDP interactions. The inhibitory effect of ATP is largely attributed to its competitive binding mode to Arg residues. Similar competitive binding of ATP is also observed in FUS PLD. Both ATP binding and the PLD interaction share the residues including Gln, Ser, and Tyr residues, while the competition is rather modest due to the abundance of these residues in the sequence. Interestingly, ATP undergoes considerable diffusion on the surface of PLD, which is an order of magnitude faster than the evolution of the contact area of PLDs. The temporal separation of these two processes remarkably promotes the inhibitory effect of ATP on PLD interaction. Given the representativeness of these two IDPs, competitive binding may serve as a general mechanism underlying ATP inhibition on IDP interactions at high physiological levels.

Unravelling intrinsically disordered and compositionally biased proteins in the cereal proteomes.

Intrinsically disordered proteins (IDPs) play key biological functions despite lacking predetermined 3D structures. They are often compositionally biased and characterized by specific amino acid compositions. Here, we investigated protein intrinsic disorder in rice, wheat, barley, maize, sorghum, oat and rye proteomes. Then, we studied the distribution of compositionally biased proteins (CBs) in these species. The data showed that the contents of compositional biased proteins (CB), the average protein sizes, and biased sequence sizes were similar in the studied proteomes. Furthermore, the CB proteins were enriched in intrinsic disorder and IDPs were characterized by noticeable composition biases. In addition, the polar and the charged residues were the most abundant among the types of the biased residues. Gene Ontology analysis revealed that CB proteins in the studied species are mainly involved in binding, catalytic activity, and transcription regulation. Altogether, our findings indicated that there is a noticeable conservation of intrinsically disordered and CB proteins in cereals. The evolutionary conservation of these features implies that cereals may use common cellular and regulatory mechanisms to adapt to various environmental constraints.

Versatile roles of disordered transcription factor effector domains in transcriptional regulation.

Transcription, a crucial step in the regulation of gene expression, is tightly controlled and involves several essential processes, such as chromatin organization, recognition of the specific genomic sequences, DNA binding, and ultimately recruiting the transcriptional machinery to facilitate transcript synthesis. At the center of this regulation are transcription factors (TFs), which comprise at least one DNA-binding domain (DBD) and an effector domain (ED). Although the structure and function of DBDs have been well studied, our knowledge of the structure and function of effector domains is limited. EDs are of particular importance in generating distinct transcriptional responses between protein members of the same TF family that have similar DBDs and specificities. The study of transcriptional activity conferred by effector domains has traditionally been conducted through examining protein-protein interactions. However, recent research has uncovered alternative mechanisms by which EDs regulate gene expression, such as the formation of condensates that increase the local concentration of transcription factors, cofactors, and coregulated genes, as well as DNA binding. Here, we provide a comprehensive overview of the known roles of transcription factor EDs, with a specific focus on disordered regions. Additionally, we emphasize the significance of intrinsically disordered regions (IDRs) during transcriptional regulation. We examine the mechanisms underlying the establishment and maintenance of transcriptional specificity through the structural properties of predominantly disordered EDs. We then provide a comprehensive overview of the current understanding of these domains, including their physical and chemical characteristics, as well as their functional roles.

USP39 phase separates into the nucleolus and drives lung adenocarcinoma progression by promoting GLI1 expression

BackgroundIntracellular membraneless organelles formed by liquid-liquid phase separation (LLPS) function in diverse physiological processes and have been linked to tumor-promoting properties. The nucleolus is one of the largest membraneless organelle formed through LLPS. Deubiquitylating enzymes (DUBs) emerge as novel therapeutic targets against human cancers. However, the nucleolar phase separation of DUBs and association with lung cancer development have remained incompletely investigated till now.MethodsGFP-USP39 fusion proteins were analyzed for LLPS properties using immunofluorescence, fluorescence recovery after photobleaching (FRAP) and in vitro LLPS assays. Intrinsically-disordered regions of USP39 were analyzed by PhaSepDB database. Transcriptomic profiling, Western blot, RT-PCR and luciferase reporter assays were conducted to identify targets regulated by USP39. The effects of USP39 depletion on tumor progression were tested using doxycycline-inducible USP39 knockdown and rescue lung adenocarcinoma cells both in vitro and in vivo by performing MTT, colony formation, EdU staining, transwell and tumor xenograft model experiments.ResultsUSP39 phase separates into nucleoli depending upon its N-terminal disordered region with amino acid residues 1-103. Lung cancer cell growth and migration were dramatically inhibited by USP39 knockdown, which was rescued by exogenous USP39 complementation. Moreover, knockdown of USP39 reduced oncogenic transcription effector GLI1 levels. Finally, USP39 downregulation restricted the formation of lung cancer xenografts in nude mice.ConclusionsUSP39 undergoes LLPS in the nucleolus and promotes tumor progression by regulating GLI1 expression. Downregulation of USP39 effectively suppressed lung cancer growth, and therefore targeting USP39 provides novel therapeutic strategy to treat lung cancer.

Genome-Wide Characterization of Wholly Disordered Proteins in Arabidopsis

Intrinsically disordered proteins (IDPs) include two types of proteins: partial disordered regions (IDRs) and wholly disordered proteins (WDPs). Extensive studies focused on the proteins with IDRs, but less is known about WDPs because of their difficult-to-form folded tertiary structure. In this study, we developed a bioinformatics method for screening more than 50 amino acids in the genome level and found a total of 27 categories, including 56 WDPs, in Arabidopsis. After comparing with 56 randomly selected structural proteins, we found that WDPs possessed a more wide range of theoretical isoelectric point (PI), a more negative of Grand Average of Hydropathicity (GRAVY), a higher value of Instability Index (II), and lower values of Aliphatic Index (AI). In addition, by calculating the FCR (fraction of charged residue) and NCPR (net charge per residue) values of each WDP, we found 20 WDPs in R1 (FCR < 0.25 and NCPR < 0.25) group, 15 in R2 (0.25 ≤ FCR ≤ 0.35 and NCPR ≤ 0.35), 19 in R3 (FCR > 0.35 and NCPR ≤ 0.35), and two in R4 (FCR > 0.35 and NCPR > 0.35). Moreover, the gene expression and protein-protein interaction (PPI) network analysis showed that WDPs perform different biological functions. We also showed that two WDPs, SIS (Salt Induced Serine rich) and RAB18 (a dehydrin family protein), undergo the in vitro liquid-liquid phase separation (LLPS). Therefore, our results provide insight into understanding the biochemical characters and biological functions of WDPs in plants.

Bioinformatics-Driven Structural and Pharmacological Analysis of SLITRK1 in Tourette Syndrome: Impact of S656M Mutation Using Molecular Dynamics, Docking, and Reinforcement Learning

Abstract: SLITRK1 is a critical protein involved in neural development and is associated with various neurological disorders, including Tourette Syndrome. This study investigates the structural dynamics, intrinsic disorder propensity, and pharmacological interactions of SLITRK1, with a particular focus on amino acid substitutions and their pathological implications. A comprehensive computational framework was employed, including intrinsic disorder region analysis, transmembrane topology predictions, and stability assessments of SLITRK1 variants. Integrated with reinforcement learning (RL), molecular docking and dynamics simulations were used to evaluate the pharmacotherapeutic potential of drugs commonly prescribed for Tourette Syndrome, such as Pimozide, Aripiprazole, Risperidone, and Haloperidol. Structural analyses revealed that the S656M mutation significantly alters SLITRK1’s 3D conformation, biological functions, and drug binding profiles. Among the tested drugs, Aripiprazole exhibited the highest binding affinity across various SLITRK1 variants, with reinforcement learning highlighting a notable interaction with the S659K mutation. These findings were supported by Ramachandran plot and molecular dynamics analyses, which identified mutation-induced structural and dynamic changes. This study provides an integrative analysis of SLITRK1, offering insights into its role in Tourette Syndrome and laying a foundation for targeted therapeutic strategies to mitigate SLITRK1-related neurological disorders.

Ensemble docking for intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are implicated in many human diseases and are increasingly being pursued as drug targets. Conventional structure-based drug design methods that rely on well-defined binding sites are however, largely unsuitable for IDPs. Here, we present computationally efficient ensemble docking approaches to predict the relative affinities of small molecules to IDPs and characterize their dynamic, heterogenous binding mechanisms at atomic resolution. We demonstrate that these ensemble docking protocols accurately predict the relative binding affinities of small molecule α-synuclein ligands measured by NMR spectroscopy and generate conformational ensembles of ligand binding modes in remarkable agreement with experimentally validated long-timescale molecular dynamics simulations. Our results display the potential of ensemble docking approaches for predicting small molecule binding to IDPs and suggest that these methods may be valuable tools for IDP drug discovery campaigns.

Implementation of Time-Averaged Restraints with UNRES Coarse-Grained Model of Polypeptide Chains.

Time-averaged restraints from nuclear magnetic resonance (NMR) measurements have been implemented in the UNRES coarse-grained model of polypeptide chains in order to develop a tool for data-assisted modeling of the conformational ensembles of multistate proteins, intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs), many of which are essential in cell biology. A numerically stable variant of molecular dynamics with time-averaged restraints has been introduced, in which the total energy is conserved in sections of a trajectory in microcanonical runs, the bath temperature is maintained in canonical runs, and the time-average-restraint-force components are scaled up with the length of the memory window so that the restraints affect the simulated structures. The new approach restores the conformational ensembles used to generate ensemble-averaged distances, as demonstrated with synthetic restraints. The approach results in a better fitting of the ensemble-averaged interproton distances to those determined experimentally for multistate proteins and proteins with intrinsically disordered regions, which puts it at an advantage over all-atom approaches with regard to the determination of the conformational ensembles of proteins with diffuse structures, owing to a faster and more robust conformational search.

Inhibition of HMGA2 binding to AT-rich DNA by its negatively charged C-terminus.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a small DNA-binding protein that specifically targets AT-rich DNA sequences. Structurally, HMGA2 is an intrinsically disordered protein (IDP), comprising three positively charged 'AT-hooks' and a negatively charged C-terminus. HMGA2 can form homodimers through electrostatic interactions between its 'AT-hooks' and C-terminus. This suggests that the negatively charged C-terminus may inhibit DNA binding by interacting with the positively charged 'AT-hooks.' In this paper, we demonstrate that the C-terminus significantly influences HMGA2's DNA-binding properties. For example, the C-terminal deletion mutant HMGA2Δ95-108 binds more tightly to the AT-rich DNA oligomer FL814 than wild-type HMGA2. Additionally, a synthetic peptide derived from the C-terminus (the C-terminal motif peptide or CTMP) strongly inhibits HMGA2's binding to FL814, likely by interacting with the 'AT-hooks,' as shown by various biochemical and biophysical assays. Molecular modeling demonstrates that electrostatic interactions and hydrogen bonding are the primary forces driving CTMP's binding to the 'AT-hooks.' Intriguingly, we found that hydration does not play a role in HMGA2-DNA binding. These results suggest that the highly negatively charged C-terminus of HMGA2 plays a critical role in regulating its DNA-binding capacity through autoinhibition, likely facilitating the target search process for AT-rich DNA sequences.

The splicing auxiliary factor OsU2AF35a enhances thermotolerance via protein separation and promoting proper splicing of OsHSA32 pre-mRNA in rice.

Heat stress significantly impacts global rice production, highlighting the critical need to understand the genetic basis of heat resistance in rice. U2AF (U2 snRNP auxiliary factor) is an essential splicing complex with critical roles in recognizing the 3'-splice site of precursor messenger RNAs (pre-mRNAs). The U2AF small subunit (U2AF35) can bind to the 3'-AG intron border and promote U2 snRNP binding to the branch-point sequences of introns through interaction with the U2AF large subunit (U2AF65). However, the functions of U2AF35 in plants are poorly understood. In this study, we discovered that the OsU2AF35a gene was vigorously induced by heat stress and could positively regulate rice thermotolerance during both the seedling and reproductive growth stages. OsU2AF35a interacts with OsU2AF65a within the nucleus, and both of them can form condensates through liquid-liquid phase separation (LLPS) following heat stress. The intrinsically disordered regions (IDR) are accountable for their LLPS. OsU2AF35a condensation is indispensable for thermotolerance. RNA-seq analysis disclosed that, subsequent to heat treatment, the expression levels of several genes associated with water deficiency and oxidative stress in osu2af35a-1 were markedly lower than those in ZH11. In accordance with this, OsU2AF35a is capable of positively regulating the oxidative stress resistance of rice. The pre-mRNAs of a considerable number of genes in the osu2af35a-1 mutant exhibited defective splicing, among which was the OsHSA32 gene. Knocking out OsHSA32 significantly reduced the thermotolerance of rice, while overexpressing OsHSA32 could partially rescue the heat sensitivity of osu2af35a-1. Together, our findings uncovered the essential role of OsU2AF35a in rice heat stress response through protein separation and regulating alternative pre-mRNA splicing.

Supramolecular Switching of Liquid-Liquid Phase Separation for Orchestrating Enzyme Kinetics.

Dynamic liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) and associated assembly and disassembly of biomolecular condensates play crucial roles in cellular organization and metabolic networks. These processes are often regulated by supramolecular interactions. However, the complex and disordered structures of IDPs, coupled with their rapid conformational fluctuations, pose significant challenges for reconstructing supramolecularly-regulated dynamic LLPS systems and quantitatively illustrating variations in molecular interactions. Inspired by the structural feature of IDPs that facilitates LLPS, we designed a simplified phase-separating molecule, Nap-o-Nap, consisting of two naphthalene moieties linked by an ethylene glycol derivative. This compound exhibits LLPS under physiological conditions, forming coacervate microdroplets that undergo multiple cycles of disassembly and reassembly upon stoichiometric addition of Cucurbit[7]uril and Adamantane, respectively, based upon competitive host-guest interactions. Importantly, such reversible control offers a unique route to quantify entropically dominant nature (ΔS= 14.0 cal∙mol-1∙K-1) within the LLPS process, in which the binding affinity of host-guest interactions (ΔG= -14.9 kcal∙mol-1) surpass that of the LLPS of Nap-o-Nap (ΔG= -2.1 kcal∙mol-1), enabling the supramolecular regulation process. The supramolecularly switched LLPS, along with selective client recruitment and exclusion by resultant coacervates, provides a promising platform for either boosting or retarding enzymatic reactions, thereby orchestrating biological enzyme kinetics.

Supramolecular Switching of Liquid‐Liquid Phase Separation for Orchestrating Enzyme Kinetics

Dynamic liquid‐liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) and associated assembly and disassembly of biomolecular condensates play crucial roles in cellular organization and metabolic networks. These processes are often regulated by supramolecular interactions. However, the complex and disordered structures of IDPs, coupled with their rapid conformational fluctuations, pose significant challenges for reconstructing supramolecularly‐regulated dynamic LLPS systems and quantitatively illustrating variations in molecular interactions. Inspired by the structural feature of IDPs that facilitates LLPS, we designed a simplified phase‐separating molecule, Nap‐o‐Nap, consisting of two naphthalene moieties linked by an ethylene glycol derivative. This compound exhibits LLPS under physiological conditions, forming coacervate microdroplets that undergo multiple cycles of disassembly and reassembly upon stoichiometric addition of Cucurbit[7]uril and Adamantane, respectively, based upon competitive host‐guest interactions. Importantly, such reversible control offers a unique route to quantify entropically dominant nature (ΔS= 14.0 cal∙mol‐1∙K‐1) within the LLPS process, in which the binding affinity of host‐guest interactions (ΔG= −14.9 kcal∙mol‐1) surpass that of the LLPS of Nap‐o‐Nap (ΔG= −2.1 kcal∙mol‐1), enabling the supramolecular regulation process. The supramolecularly switched LLPS, along with selective client recruitment and exclusion by resultant coacervates, provides a promising platform for either boosting or retarding enzymatic reactions, thereby orchestrating biological enzyme kinetics.

Conformationally adaptive therapeutic peptides for diseases caused by intrinsically disordered proteins (IDPs). New paradigm for drug discovery: Target the target, not the arrow.

The traditional model of protein structure determined by the amino acid sequence is today seriously challenged by the fact that approximately half of the human proteome is made up of proteins that do not have a stable 3D structure, either partially or in totality. These proteins, called intrinsically disordered proteins (IDPs), are involved in numerous physiological functions and are associated with severe pathologies, e.g. Alzheimer, Parkinson, Creutzfeldt-Jakob, amyotrophic lateral sclerosis (ALS), and type 2 diabetes. Targeting these proteins is challenging for two reasons: i) we need to preserve their physiological functions, and ii) drug design by molecular docking is not possible due to the lack of reliable starting conditions. Faced with this challenge, the solutions proposed by artificial intelligence (AI) such as AlphaFold are clearly unsuitable. Instead, we suggest an innovative approach consisting of mimicking, in short synthetic peptides, the conformational flexibility of IDPs. These peptides, which we call adaptive peptides, are derived from the domains of IDPs that become structured after interacting with a ligand. Adaptive peptides are designed with the aim of selectively antagonizing the harmful effects of IDPs, without targeting them directly but through selected ligands, without affecting their physiological properties. This "target the target, not the arrow" strategy is promised to open a new route to drug discovery for currently undruggable proteins.

Microexon in action: How tiny fragments in a protein tune function, drive disease.

Intrinsically disordered regions (IDRs) of proteins can regulate function through phase separation. In a recent article in Nature, Garcia-Cabau etal. reveal that including or excluding a microexon within the IDR of CPEB4 alters its condensation properties, suggesting a potential mechanism underlying autism spectrum disorder.1.

Targeting FOXM1 condensates reduces breast tumour growth and metastasis.

Identifying phase-separated structures remains challenging, and effective intervention methods are currently lacking1. Here we screened for phase-separated proteins in breast tumour cells and identified forkhead (FKH) box protein M1 (FOXM1) as the most prominent candidate. Oncogenic FOXM1 underwent liquid-liquid phase separation (LLPS) with FKH consensus DNA element, and compartmentalized the transcription apparatus in the nucleus, thereby sustaining chromatin accessibility and super-enhancer landscapes crucial for tumour metastatic outgrowth. Screening an epigenetics compound library identified AMPK agonists as suppressors of FOXM1 condensation. AMPK phosphorylated FOXM1 in the intrinsically disordered region (IDR), perturbing condensates, reducing oncogenic transcription, accumulating double-stranded DNA to stimulate innate immune responses, and endowing discrete FOXM1 with the ability to activate immunogenicity-related gene expressions. By developing a genetic code-expansion orthogonal system, we demonstrated that a phosphoryl moiety at a specific IDR1 site causes electrostatic repulsion, thereby abolishing FOXM1 LLPS and aggregation. A peptide targeting IDR1 and carrying the AMPK-phosphorylated residue was designed to disrupt FOXM1 LLPS and was shown to inhibit tumour malignancy, rescue tumour immunogenicity and improve tumour immunotherapy. Together, these findings provide novel and in-depth insights on function and mechanism of FOXM1 and develop methodologies that hold promising implications in clinics.

Scale-free Spatio-temporal Correlations in Conformational Fluctuations of Intrinsically Disordered Proteins.

The self-assembly of intrinsically disordered proteins (IDPs) into condensed phases and the formation of membrane-less organelles (MLOs) can be considered as the phenomenon of collective behavior. The conformational dynamics of IDPs are essential for their interactions and the formation of a condensed phase. From a physical perspective, collective behavior and the emergence of phase are associated with long-range correlations. Here the conformational dynamics of IDPs and the correlations therein are analyzed, using µs-scale atomistic molecular dynamics (MD) simulations and single-molecule Förster resonance energy transfer (smFRET) experiments. The existence of typical scale-free spatio-temporal correlations in IDP conformational fluctuations is demonstrated. Their conformational evolutions exhibit "1/f noise" power spectra and are accompanied by the appearance of residue domains following a power-law size distribution. Additionally, the motions of residues present scale-free behavioral correlation. These scale-free correlations resemble those in physical systems near critical points, suggesting that IDPs are poised at a critical state. Therefore, IDPs can effectively respond to finite differences in sequence compositions and engender considerable structural heterogeneity which is beneficial for IDP interactions and phase formation.

Context-Dependent Heterotypic Assemblies of Intrinsically Disordered Peptides.

Despite their critical role in context-dependent interactions for protein functions, intrinsically disordered regions (IDRs) are often overlooked for designing peptide assemblies. Here, we exploit IDRs to enable context-dependent heterotypic assemblies of intrinsically disordered peptides, where "context-dependent" refers to assembly behavior driven by interactions with other molecules. By attaching an aromatic segment to oppositely charged intrinsically disordered peptides, we achieve a nanofiber formation. Although the same-charged peptides cannot self-assemble, oppositely charged peptides form heterotypic nanofibers. Cryo-EM analysis reveals a β-sheet arrangement within the ordered core of these nanofibers, conformational heterogeneity, and a disorder-to-order continuum and shows a high number of hydrogen bonds between tyrosine and lysine ε-amine. Additionally, this work demonstrates a post-assembly morphological change resulting from local conformational flexibility. While equal molar mixtures of the charged intrinsically disordered peptides yield nanofibers, doubling the positively charged peptides after assembly produces bundles of nanofibers. Furthermore, reducing the number of aromatic amino acid residues reduces bundle formation. Demonstrating context-dependent self-assembly of intrinsically disordered peptides and revealing atomistic insights into heterotypic assemblies of intrinsically disordered peptides for the first time, this work illustrates a straightforward approach to enable heterotypic intrinsically disordered peptides to self-assemble for the design of adaptive, multifunctional peptide nanomaterials.