Intrinsically Disordered Proteins Research Articles

Effects of Aging on Intrinsic Protein Disorder in Human Lenses and Zonules.

This study aims to compare the levels of intrinsic protein disorder within the human lens and zonule proteomes and investigate the role of aging as a potential influencing factor on disorder levels. A cross-sectional proteomic analysis was employed, utilizing a dataset of 1466 proteins derived from the lens and zonule proteomes previously published by Wang et al. and De Maria et al. Bioinformatics tools, including a composition profiler and a rapid intrinsic disorder analysis online tool, were used to conduct a comparative analysis of protein disorder. Statistical tests such as ANOVA, Tukey's HSD, and chi-squared tests were applied to evaluate differences between groups. The study revealed distinct amino acid compositions for each proteome, showing a direct correlation between aging and increased protein disorder in the zonular proteomes, whereas the lens proteomes exhibited the opposite trend. Findings suggest that age-related changes in intrinsic protein disorder within the lens and zonule proteomes may be linked to structural transformations in these tissues. Understanding how protein disorder evolves with age could enhance knowledge of the molecular basis for age-related conditions such as cataracts and pseudoexfoliation, potentially leading to better therapeutic strategies.

Differential Effects of Sequence-Local versus Nonlocal Charge Patterns on Phase Separation and Conformational Dimensions of Polyampholytes as Model Intrinsically Disordered Proteins.

Conformational properties of intrinsically disordered proteins (IDPs) are governed by a sequence-ensemble relationship. To differentiate the impact of sequence-local versus sequence-nonlocal features of an IDP's charge pattern on its conformational dimensions and its phase-separation propensity, the charge "blockiness" κ and the nonlocality-weighted sequence charge decoration (SCD) parameters are compared for their correlations with isolated-chain radii of gyration (Rgs) and upper critical solution temperatures (UCSTs) of polyampholytes modeled by random phase approximation, field-theoretic simulation, and coarse-grained molecular dynamics. SCD is superior to κ in predicting Rg because SCD accounts for effects of contact order, i.e., nonlocality, on dimensions of isolated chains. In contrast, κ and SCD are comparably good, though nonideal, predictors of UCST because frequencies of interchain contacts in the multiple-chain condensed phase are less sensitive to sequence positions than frequencies of intrachain contacts of an isolated chain, as reflected by κ correlating better with condensed-phase interaction energy than SCD.

Translational Diffusion and Self-Association of an Intrinsically Disordered Protein κ-Casein Using NMR with Ultra-High Pulsed-Field Gradient and Time-Resolved FRET.

Much attention has been given to studying the translational diffusion of globular proteins, whereas the translational diffusion of intrinsically disordered proteins (IDPs) is less studied. In this study, we investigate the translational diffusion and how it is affected by the self-association of an IDP, κ-casein, using pulsed-field gradient nuclear magnetic resonance and time-resolved Förster resonance energy transfer. Using the analysis of the shape of diffusion attenuation and the concentration dependence of κ-casein diffusion coefficients and intermolecular interactions, we demonstrate that κ-casein exhibits continuous self-association. When the volume fraction of κ-casein is below 0.08, we observe that κ-casein self-association results in a macroscopic phase separation upon storage at 4 °C. At κ-casein volume fractions above 0.08, self-association leads to the formation of labile gel-like networks without subsequent macroscopic phase separation. Unlike α-casein, which shows a strong concentration dependence and extensive gel-like network formation, only one-third of κ-casein molecules participate in the gel network at a time, resulting in a more dynamic and less extensive structure. These findings highlight the unique association properties of κ-casein, contributing to a better understanding of its behavior under various conditions and its potential role in casein micelle formation.

Streamlining NMR Chemical Shift Predictions for Intrinsically Disordered Proteins: Design of Ensembles with Dimensionality Reduction and Clustering.

By merging advanced dimensionality reduction (DR) and clustering algorithm (CA) techniques, our study advances the sampling procedure for predicting NMR chemical shifts (CS) in intrinsically disordered proteins (IDPs), making a significant leap forward in the field of protein analysis/modeling. We enhance NMR CS sampling by generating clustered ensembles that accurately reflect the different properties and phenomena encapsulated by the IDP trajectories. This investigation critically assessed different rapid CS predictors, both neural network (e.g., Sparta+ and ShiftX2) and database-driven (ProCS-15), and highlighted the need for more advanced quantum calculations and the subsequent need for more tractable-sized conformational ensembles. Although neural network CS predictors outperformed ProCS-15 for all atoms, all tools showed poor agreement with HN CSs, and the neural network CS predictors were unable to capture the influence of phosphorylated residues, highly relevant for IDPs. This study also addressed the limitations of using direct clustering with collective variables, such as the widespread implementation of the GROMOS algorithm. Clustered ensembles (CEs) produced by this algorithm showed poor performance with chemical shifts compared to sequential ensembles (SEs) of similar size. Instead, we implement a multiscale DR and CA approach and explore the challenges and limitations of applying these algorithms to obtain more robust and tractable CEs. The novel feature of this investigation is the use of solvent-accessible surface area (SASA) as one of the fingerprints for DR alongside previously investigated α carbon distance/angles or ϕ/ψ dihedral angles. The ensembles produced with SASA tSNE DR produced CEs better aligned with the experimental CS of between 0.17 and 0.36 r2 (0.18-0.26 ppm) depending on the system and replicate. Furthermore, this technique produced CEs with better agreement than traditional SEs in 85.7% of all ensemble sizes. This study investigates the quality of ensembles produced based on different input features, comparing latent spaces produced by linear vs nonlinear DR techniques and a novel integrated silhouette score scanning protocol for tSNE DR.

Evolutionary analysis of ZAP and its cofactors identifies intrinsically disordered regions as central elements in host-pathogen interactions

The zinc-finger antiviral protein (ZAP) is an innate immunity sensor of non-self nucleic acids. Its antiviral activity is exerted through the physical interaction with different cofactors, including TRIM25, Riplet and KHNYN. Cellular proteins that interact with infectious agents are expected to be engaged in genetic conflicts that often result in their rapid evolution. To test this possibility and to identify the regions most strongly targeted by natural selection, we applied in silico molecular evolution tools to analyze the evolutionary history of ZAP and cofactors in four mammalian groups. We report evidence of positive selection in all genes and in most mammalian groups. On average, the intrinsically disordered regions (IDRs) embedded in the four proteins evolve significantly faster than folded domains and most positively selected sites fall within IDRs. In ZAP, the PARP domain also shows abundant signals of selection, and independent evolution in different mammalian groups suggests modulation of its ADP-ribose binding ability. Detailed analyses of the biophysical properties of IDRs revealed that chain compaction and conformational entropy are conserved across mammals. The IDRs in ZAP and KHNYN are particularly compact, indicating that they may promote phase separation (PS). In line with this hypothesis, we predicted several PS-promoting regions in ZAP and KHNYN, as well as in TRIM25. Positively selected sites are abundant in these regions, suggesting that PS may be important for the antiviral functions of these proteins and the evolutionary arms race with viruses. Our data shed light into the evolution of ZAP and cofactors and indicate that IDRs represent central elements in host-pathogen interactions.

Disordered proteins interact with the chemical environment to tune their protective function during drying.

The conformational ensemble and function of intrinsically disordered proteins (IDPs) are sensitive to their solution environment. The inherent malleability of disordered proteins combined with the exposure of their residues accounts for this sensitivity. One context in which IDPs play important roles that is concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has long been linked to the accumulation of high levels of cosolutes such as trehalose or sucrose as well as the enrichment of IDPs, such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat soluble (CAHS) proteins. Despite knowing that IDPs play important roles and are co-enriched alongside endogenous, species-specific cosolutes during desiccation, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance. Here, we test the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by endogenous cosolutes. We find that desiccation-related IDPs derived from four different organisms spanning two LEA protein families and the CAHS protein family, synergize best with endogenous cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. We further demonstrate that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel. Our results suggest that functional synergy between IDPs and endogenous cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet, the mechanisms underlying this synergy differ between IDP families.

Characterization of Intrinsically Disordered Proteins in Healthy and Diseased States by Nuclear Magnetic Resonance.

Intrinsically Disordered Proteins (IDPs) are active in different cellular procedures like ordered assembly of chromatin and ribosomes, interaction with membrane, protein, and ligand binding, molecular recognition, binding, and transportation via nuclear pores, microfilaments and microtubules process and disassembly, protein functions, RNA chaperone, and nucleic acid binding, modulation of the central dogma, cell cycle, and other cellular activities, post-translational qualification and substitute splicing, and flexible entropic linker and management of signaling pathways. The intrinsic disorder is a precise structural characteristic that permits IDPs/IDPRs to be involved in both one-to-many and many-to-one signaling. IDPs/IDPRs also exert some dynamical and structural ordering, being much less constrained in their activities than folded proteins. Nuclear magnetic resonance (NMR) spectroscopy is a major technique for the characterization of IDPs, and it can be used for dynamic and structural studies of IDPs. This review was carried out to discuss intrinsically disordered proteins and their different goals, as well as the importance and effectiveness of NMR in characterizing intrinsically disordered proteins in healthy and diseased states.

Intrinsic disorder and salt-dependent conformational changes of the N-terminal region of TFIP11 splicing factor

Tuftelin Interacting Protein 11 (TFIP11) was identified as a critical human spliceosome assembly regulator, interacting with multiple proteins and localising in membrane-less organelles. However, a lack of structural information on TFIP11 limits the rationalisation of its biological role. TFIP11 is predicted as an intrinsically disordered protein (IDP), and more specifically concerning its N-terminal (N-TER) region. IDPs lack a defined tertiary structure, existing as a dynamic conformational ensemble, favouring protein-protein and protein-RNA interactions. IDPs are involved in liquid-liquid phase separation (LLPS), driving the formation of subnuclear compartments.Combining disorder prediction, molecular dynamics, and spectroscopy methods, this contribution shows the first evidence TFIP11 N-TER is a polyampholytic IDP, exhibiting a structural duality with the coexistence of ordered and disordered assemblies, depending on the ionic strength. Increasing the salt concentration enhances the protein conformational flexibility, presenting a more globule-like shape, and a fuzzier unstructured arrangement that could favour LLPS and protein-RNA interaction. The most charged and hydrophilic regions are the most impacted, including the G-Patch domain essential to TFIP11 function.This study gives a better understanding of the salt-dependent conformational behaviour of the N-TER TFIP11, supporting the hypothesis of the formation of different types of protein assembly, in line with its multiple biological roles.

Autoregulatory mechanism of enzyme activity by the nuclear localization signal of lysine-specific demethylase 1

The N-terminal region of the human Lysine Specific Demethylase 1 (LSD1) has no predicted structural elements, contains a nuclear localization signal (NLS), undergoes multiple post-translational modifications (PTMs), and acts as a protein-protein interaction hub. This intrinsically disordered region (IDR) extends from core LSD1 structure, resides atop the catalytic active site, and is known to be dispensable for catalysis. Here, we show differential nucleosome binding between the full-length and an N-terminus deleted LSD1 and identify that a conserved NLS and PTM containing element of the N-terminus contains an alpha helical structure, and that this conserved element impacts demethylation. Enzyme assays reveal that LSD1’s own electropositive NLS amino acids 107-120 inhibit demethylation activity on a model Histone 3 lysine 4 di-methyl (H3K4me2) peptide (Kiapp ∼ 3.3 μM) and H3K4me2 nucleosome substrates (IC50 ∼ 30.4 μM), likely mimicking the histone H3 tail. Further, when the identical, inhibitory NLS region contains phosphomimetic modifications, inhibition is partially relieved. Based upon these results and biophysical data, a regulatory mechanism for the LSD1-catalyzed demethylation reaction is proposed whereby NLS-mediated autoinhibition can occur through electrostatic interactions, and be partially relieved through phosphorylation that occurs proximal to the NLS. Taken together, the results highlight a dynamic and synergistic role for PTMs, IDRs, and structured regions near LSD1 active site and introduces the notion that phosphorylated mediated NLS regions can function to fine-tune chromatin modifying enzyme activity.

Hydrogen Bonding-Driven Self-Coacervation of Nonionic Homopolymers for Stimuli-Triggered Therapeutic Release.

Inspired by the unique functionalities of biomolecular membraneless organelles (MLOs) formed via liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) and nucleic acids, a great deal of effort has been devoted to devising phase-separated artificial subcellular dynamic compartments. These endeavors aim to unravel the molecular mechanism underlying the formation and intracellular delivery of susceptible macromolecular therapeutics. We report herein pyroglutamic acid (PGA)-based well-defined homopolymers featuring stimuli-tunable reversible self-coacervation ability. The polymer exhibits an upper critical solution temperature (UCST) transition in aqueous solutions and has the propensity to undergo cooling-induced LLPS, producing micrometer-sized liquid droplets. This phase separation phenomenon could be modulated by various factors, including polymer concentration, chain length, solution pH, and types and concentrations of different additives. These micrometer droplets are thermally reversible and encapsulate a wide variety of cargoes, including small hydrophobic fluorescent molecules, hydrophilic anticancer drugs, and fluorophore-labeled macromolecular proteins (bovine serum albumin and lysozyme). The payloads were released by exploiting the thermo/pH-mediated disassembly behavior of the coacervates, preserving the bioactivity of the sensitive therapeutics. This environmentally responsive, simple yet versatile artificial MLO model system will provide insights into the biomolecular nonionic condensates and pave the way for the de novo design of dynamic biomolecule depots.

Impact of protein domains on the MEL2 granule, a cytoplasmic ribonucleoprotein complex maintaining faithful meiosis progression in rice.

Cytoplasmic ribonucleoprotein (RNP) granules are membraneless structures composed of various RNAs and proteins that play important roles in post-transcriptional regulation. While RNP granules are known to regulate the meiotic entry in some organisms, little is known about their roles in plants. In this study, we observed the cytoplasmic granular structures of rice RNA-binding protein MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2), which contributes to the control of meiotic entry timing, in leaf protoplasts and spore mother cells. We performed colocalization analysis with known cytoplasmic RNP factors, and domain deletion analysis to assess their impact on granule formation and meiosis progression. Conservation of MEL2 domains across plant species was also explored. Our results indicated that MEL2 granules colocalized with processing body and stress granule factors. The maintenance of granule properties modulated by LOTUS domain and the intrinsically disordered region (IDR) is essential for proper MEL2 function in meiosis progression. MEL2-like proteins widely found in plant kingdom conserved LOTUS domain followed by the IDR despite their diverse domain structures, suggesting the functional conservation of these domains among plant species. This study highlights the role of MEL2 granule dynamics and its impact on meiotic transition and progression.

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.