The dual life of disordered lysine-rich domains of snoRNPs in rRNA modification and nucleolar compaction.

Intrinsically disordered regions (IDRs) are highly enriched in the nucleolar proteome but their physiological role in ribosome assembly remains poorly understood. Our study reveals the functional plasticity of the extremely abundant lysine-rich IDRs of small nucleolar ribonucleoprotein particles (snoRNPs) from protists to mammalian cells. We show in Saccharomyces cerevisiae that the electrostatic properties of this lysine-rich IDR, the KKE/D domain, promote snoRNP accumulation in the vicinity of nascent rRNAs, facilitating their modification. Under stress conditions reducing the rate of ribosome assembly, they are essential for nucleolar compaction and sequestration of key early-acting ribosome biogenesis factors, including RNA polymerase I, owing to their self-interaction capacity in a latent, non-rRNA-associated state. We propose that such functional plasticity of these lysine-rich IDRs may represent an ancestral eukaryotic regulatory mechanism, explaining how nucleolar morphology is continuously adapted to rRNA production levels.

Disordered regions of human eIF4B orchestrate a dynamic self-association landscape.

Eukaryotic translation initiation factor eIF4B is required for efficient cap-dependent translation, it is overexpressed in cancer cells, and may influence stress granule formation. Due to the high degree of intrinsic disorder, eIF4B is rarely observed in cryo-EM structures of translation complexes and only ever by its single structured RNA recognition motif domain, leaving the molecular details of its large intrinsically disordered region (IDR) unknown. By integrating experiments and simulations we demonstrate that eIF4B IDR orchestrates and fine-tunes an intricate transition from monomers to a condensed phase, in which large-size dynamic oligomers form before mesoscopic phase separation. Single-molecule spectroscopy combined with molecular simulations enabled us to characterize the conformational ensembles and underlying intra- and intermolecular dynamics across the oligomerization transition. The observed sensitivity to ionic strength and molecular crowding in the self-association landscape suggests potential regulation of eIF4B nanoscopic and mesoscopic behaviors such as driven by protein modifications, binding partners or changes to the cellular environment.

Structural study of the intrinsically disordered tardigrade damage suppressor protein (Dsup) and its complex with DNA.

Studies of proteins, found in one of the most stress-resistant animals tardigrade Ramazzottius varieornatus, aim to reveal molecular principles of extreme tolerance to various types of stress and developing applications based on them for medicine, biotechnology, pharmacy, and space research. Tardigrade DNA/RNA-binding damage suppressor protein (Dsup) reduces DNA damage caused by reactive oxygen spices (ROS) produced upon irradiation and oxidative stresses in Dsup-expressing transgenic organisms. This work is focused on the determination of structural features of Dsup protein and Dsup-DNA complex, which refines details of protective mechanism. For the first time, intrinsically disordered nature of Dsup protein with highly flexible structure was experimentally proven and characterized by the combination of small angle X-ray scattering (SAXS) technique, circular dichroism spectroscopy, and computational methods. Low resolution models of Dsup protein and an ensemble of conformations were presented. In addition, we have shown that Dsup forms fuzzy complex with DNA.

Self-association and multimer formation in AtLEA4-5, a desiccation-induced intrinsically disordered protein from plants.

During seed maturation, plants may experience severe desiccation, leading to the accumulation of late embryogenesis abundant (LEA) proteins. These intrinsically disordered proteins also accumulate in plant tissues under water deficit. Functional roles of LEA proteins have been proposed based on in vitro studies, where monomers are considered as the functional units. However, the potential formation of homo-oligomers has been little explored. In this work, we investigated the potential self-association of Arabidopsis thaliana group 4 LEA proteins (AtLEA4) using in vitro and in vivo approaches. LEA4 proteins represent a compelling case of study due to their high conservation throughout the plant kingdom. This protein family is characterized by a conserved N-terminal region, with a high alpha-helix propensity and invitro protective activity, as compared to the highly disordered and low-conserved C-terminal region. Our findings revealed that full-length AtLEA4 proteins oligomerize and that both terminal regions are sufficient for self-association in vitro. However, the ability of both amino and carboxy regions of AtLEA4-5 to self-associate invivo is significantly lower than that of the entire protein. Using high-resolution and quantitative fluorescence microscopy, we were able to disclose the unreported ability of LEA proteins to form high-order oligomers in planta. Additionally, we found that high-order complexes require the simultaneous engagement of both terminal regions, indicating that the entire protein is needed to attain such structural organization. This research provides valuable insights into the self-association of LEA proteins in plants and emphasizes the role of protein oligomer formation.

iDLB-Pred: identification of disordered lipid binding residues in protein sequences using convolutional neural network.

Proteins, nucleic acids, and lipids all interact with intrinsically disordered protein areas. Lipid-binding regions are involved in a variety of biological processes as well as a number of human illnesses. The expanding body of experimental evidence for these interactions and the dearth of techniques to anticipate them from the protein sequence serve as driving forces. Although large-scale laboratory techniques are considered to be essential for equipment for studying binding residues, they are time consuming and costly, making it challenging for researchers to predict lipid binding residues. As a result, computational techniques are being looked at as a different strategy to overcome this difficulty. To predict disordered lipid-binding residues (DLBRs), we proposed iDLB-Pred predictor utilizing benchmark dataset to compute feature through extraction techniques to identify relevant patterns and information. Various classification techniques, including deep learning methods such as Convolutional Neural Networks (CNNs), Deep Neural Networks (DNNs), Multilayer Perceptrons (MLPs), Recurrent Neural Networks (RNNs), Long Short-Term Memory (LSTM) networks, and Gated Recurrent Units (GRUs), were employed for model training. The proposed model, iDLB-Pred, was rigorously validated using metrics such as accuracy, sensitivity, specificity, and Matthew's correlation coefficient. The results demonstrate the predictor's exceptional performance, achieving accuracy rates of 81% on an independent dataset and 86% in 10-fold cross-validation.

AlphaFold-Multimer accurately captures interactions and dynamics of intrinsically disordered protein regions.

Interactions mediated by intrinsically disordered protein regions (IDRs) pose formidable challenges in structural characterization. IDRs are highly versatile, capable of adopting diverse structures and engagement modes. Motivated by recent strides in protein structure prediction, we embarked on exploring the extent to which AlphaFold-Multimer can faithfully reproduce the intricacies of interactions involving IDRs. To this end, we gathered multiple datasets covering the versatile spectrum of IDR binding modes and used them to probe AlphaFold-Multimer's prediction of IDR interactions and their dynamics. Our analyses revealed that AlphaFold-Multimer is not only capable of predicting various types of bound IDR structures with high success rate, but that distinguishing true interactions from decoys, and unreliable predictions from accurate ones is achievable by appropriate use of AlphaFold-Multimer's intrinsic scores. We found that the quality of predictions drops for more heterogeneous, fuzzy interaction types, most likely due to lower interface hydrophobicity and higher coil content. Notably though, certain AlphaFold-Multimer scores, such as the Predicted Aligned Error and residue-ipTM, are highly correlated with structural heterogeneity of the bound IDR, enabling clear distinctions between predictions of fuzzy and more homogeneous binding modes. Finally, our benchmarking revealed that predictions of IDR interactions can also be successful when using full-length proteins, but not as accurate as with cognate IDRs. To facilitate identification of the cognate IDR of a given partner, we established "minD," which pinpoints potential interaction sites in a full-length protein. Our study demonstrates that AlphaFold-Multimer can correctly identify interacting IDRs and predict their mode of engagement with a given partner.

Intrinsically disordered region amplifies membrane remodeling to augment selective ER-phagy.

Intrinsically disordered regions (IDRs) play a pivotal role in organellar remodeling. They transduce signals across membranes, scaffold signaling complexes, and mediate vesicular traffic. Their functions are regulated by constraining conformational ensembles through specific intra- and intermolecular interactions, physical tethering, and posttranslational modifications. The endoplasmic reticulum (ER)-phagy receptor FAM134B/RETREG1, known for its reticulon homology domain (RHD), includes a substantial C-terminal IDR housing the LC3 interacting motif. Beyond engaging the autophagic machinery, the function of the FAM134B-IDR is unclear. Here, we investigate the characteristics of the FAM134B-IDR by extensive modeling and molecular dynamics simulations. We present detailed structural models for the IDR, mapping its conformational landscape in solution and membrane-anchored configurations. Our analysis reveals that depending on the membrane anchor, the IDRs collapse onto the membrane and induce positive membrane curvature to varying degrees. The charge patterns underlying this Janus-like behavior are conserved across other ER-phagy receptors. We found that IDRs alone are sufficient to sense curvature. When combined with RHDs, they intensify membrane remodeling and drive efficient protein clustering, leading to faster budding, thereby amplifying RHD remodeling functions. Our simulations provide a perspective on IDRs of FAM134B, their Janus-like membrane interactions, and the resulting modulatory functions during large-scale ER remodeling.

Structural basis of the human transcriptional Mediator regulated by its dissociable kinase module.

The eukaryotic transcriptional Mediator comprises a large core (cMED) and a dissociable CDK8 kinase module (CKM). cMED recruits RNA polymerase II (RNA Pol II) and promotes pre-initiation complex formation in a manner repressed by the CKM through mechanisms presently unknown. Herein, we report cryoelectron microscopy structures of the complete human Mediator and its CKM. The CKM binds to multiple regions on cMED through both MED12 and MED13, including a large intrinsically disordered region (IDR) in the latter. MED12 and MED13 together anchor the CKM to the cMED hook, positioning CDK8 downstream and proximal to the transcription start site. Notably, the MED13 IDR obstructs the recruitment of RNA Pol II/MED26 onto cMED by direct occlusion of their respective binding sites, leading to functional repression of cMED-dependent transcription. Combined with biochemical and functional analyses, these structures provide a conserved mechanistic framework to explain the basis for CKM-mediated repression of cMED function.

Wnt target gene activation requires ß-catenin separation into biomolecular condensates.

The Wnt/ß-catenin signaling pathway plays numerous essential roles in animal development and tissue/stem cell maintenance. The activation of genes regulated by Wnt/ß-catenin signaling requires the nuclear accumulation of ß-catenin, a transcriptional co-activator. ß-catenin is recruited to many Wnt-regulated enhancers through direct binding to T-cell factor/lymphoid enhancer factor (TCF/LEF) family transcription factors. ß-catenin has previously been reported to form phase-separated biomolecular condensates (BMCs), which was implicated as a component of ß-catenin's mechanism of action. This function required aromatic amino acid residues in the intrinsically disordered regions (IDRs) at the N- and C-termini of the protein. In this report, we further explore a role for ß-catenin BMCs in Wnt target gene regulation. We find that ß-catenin BMCs are miscible with LEF1 BMCs in vitro and in cultured cells. We characterized a panel of ß-catenin mutants with different combinations of aromatic residue mutations in human cell culture and Drosophila melanogaster. Our data support a model in which aromatic residues across both IDRs contribute to BMC formation and signaling activity. Although different Wnt targets have different sensitivities to loss of ß-catenin's aromatic residues, the activation of every target examined was compromised by aromatic substitution. These mutants are not defective in nuclear import or co-immunoprecipitation with several ß-catenin binding partners. In addition, residues in the N-terminal IDR with no previously known role in signaling are clearly required for the activation of various Wnt readouts. Consistent with this, deletion of the N-terminal IDR results in a loss of signaling activity, which can be rescued by the addition of heterologous IDRs enriched in aromatic residues. Overall, our work supports a model in which the ability of ß-catenin to form biomolecular condensates in the nucleus is tightly linked to its function as a transcriptional co-regulator.

ECT2 peptide sequences outside the YTH domain regulate its m6A-RNA binding.

The m6A epitranscriptomic mark is the most abundant and widespread internal RNA chemical modification, which through the control of RNA acts as an important factor of eukaryote reproduction, growth, morphogenesis and stress response. The main m6A readers constitute a super family of proteins with hundreds of members that share a so-called YTH RNA binding domain. The majority of YTH proteins carry no obvious additional domain except for an Intrinsically Disordered Region (IDR). In Arabidopsis thaliana IDRs are important for the functional specialization among the different YTH proteins, known as Evolutionarily Conserved C-Terminal region, ECT 1 to 12. Here by studying the ECT2 protein and using an in vitro biochemical characterization, we show that full-length ECT2 and its YTH domain alone have a distinct ability to bind m6A, conversely to previously characterized YTH readers. We identify peptide regions outside of ECT2 YTH domain, in the N-terminal IDR, that regulate its binding to m6A-methylated RNA. Furthermore, we show that the selectivity of ECT2 binding for m6A is enhanced by a high uridine content within its neighbouring sequence, where ECT2 N-terminal IDR is believed to contact the target RNA in vivo. Finally, we also identify small structural elements, located next to ECT2 YTH domain and conserved in a large set of YTH proteins, that enhance its binding to m6A-methylated RNA. We propose from these findings that some of these regulatory regions are not limited to ECT2 or YTH readers of flowering plants but may be widespread among eukaryotic YTH readers.

Time-resolved NMR detection of prolyl-hydroxylation in intrinsically disordered region of HIF-1α.

Prolyl-hydroxylation is an oxygen-dependent posttranslational modification (PTM) that is known to regulate fibril formation of collagenous proteins and modulate cellular expression of hypoxia-inducible factor (HIF) α subunits. However, our understanding of this important but relatively rare PTM has remained incomplete due to the lack of biophysical methodologies that can directly measure multiple prolyl-hydroxylation events within intrinsically disordered proteins. Here, we describe a real-time 13C-direct detection NMR-based assay for studying the hydroxylation of two evolutionarily conserved prolines (P402 and P564) simultaneously in the intrinsically disordered oxygen-dependent degradation domain of hypoxic-inducible factor 1α by exploiting the "proton-less" nature of prolines. We show unambiguously that P564 is rapidly hydroxylated in a time-resolved manner while P402 hydroxylation lags significantly behind that of P564. The differential hydroxylation rate was negligibly influenced by the binding affinity to prolyl-hydroxylase enzyme, but rather by the surrounding amino acid composition, particularly the conserved tyrosine residue at the +1 position to P564. These findings support the unanticipated notion that the evolutionarily conserved P402 seemingly has a minimal impact in normal oxygen-sensing pathway.

Deciphering the intrinsically disordered characteristics of the FG-Nups through the lens of polymer physics.

The nuclear pore complex (NPC) is a critical gateway regulating molecular transport between the nucleus and cytoplasm. It allows small molecules to pass freely, while larger molecules require nuclear transport receptors to traverse the barrier. This selective permeability is maintained by phenylalanine-glycine-rich nucleoporins (FG-Nups), intrinsically disordered proteins that fill the NPC's central channel. The disordered and flexible nature of FG-Nups complicates their spatial characterization with conventional structural biology techniques. To address this challenge, polymer physics offers a valuable framework for describing FG-Nup behavior, reducing their complex structures to a few key parameters. In this review, we explore how polymer physics models FG-Nups using these parameters and discuss experimental efforts to quantify them in various contexts, providing insights into the conformational properties of FG-Nups.

Transcription regulation through selective partitioning: Weak interactions with a strong foundation.

In this issue of Molecular Cell, De La Cruz, Pradhan, Veettil et al.1 examine how selective partitioning of proteins via low-affinity IDR-dependent interactions may help regulate RNA polymerase II (RNA Pol II) function and identify sequence features that drive partitioning in cells.

Disorder-mediated interactions target proteins to specific condensates.

Selective compartmentalization of cellular contents is fundamental to the regulation of biochemistry. Although membrane-bound organelles control composition by using a semi-permeable barrier, biomolecular condensates rely on interactions among constituents to determine composition. Condensates are formed by dynamic multivalent interactions, often involving intrinsically disordered regions (IDRs) of proteins, yet whether distinct compositions can arise from these dynamic interactions is not known. Here, by comparative analysis of proteins differentially partitioned by two different condensates, we find that distinct compositions arise through specific IDR-mediated interactions. The IDRs of differentially partitioned proteins are necessary and sufficient for selective partitioning. Distinct sequence features are required for IDRs to partition, and swapping these sequence features changes the specificity of partitioning. Swapping whole IDRs retargets proteins and their biochemical activity to different condensates. Our results demonstrate that IDR-mediated interactions can target proteins to specific condensates, enabling the spatial regulation of biochemistry within the cell.

flDPnn2: Accurate and Fast Predictor of Intrinsic Disorder in Proteins.

Prediction of the intrinsic disorder in protein sequences is an active research area, with well over 100 predictors that were released to date. These efforts are motivated by the functional importance and high levels of abundance of intrinsic disorder, combined with relatively low amounts of experimental annotations. The disorder predictors are periodically evaluated by independent assessors in the Critical Assessment of protein Intrinsic Disorder prediction (CAID) experiments. The recently completed CAID2 experiment assessed close to 40 state-of-the-art methods demonstrating that some of them produce accurate results. In particular, flDPnn2 method, which is the successor of flDPnn that performed well in the CAID1 experiment, secured the overall most accurate results on the Disorder-NOX dataset in CAID2. flDPnn2 implements a number of improvements when compared to its predecessor including changes to the inputs, increased size of the deep network model that we retrained on a larger training set, and addition of an alignment module. Using results from CAID2, we show that flDPnn2 produces accurate predictions very quickly, modestly improving over the accuracy of flDPnn and reducing the runtime by half, to about 27 s per protein. flDPnn2 is freely available as a convenient web server at http://biomine.cs.vcu.edu/servers/flDPnn2/.

Replication licensing regulated by a short linear motif within an intrinsically disordered region of origin recognition complex.

In eukaryotes, the origin recognition complex (ORC) faciliates the assembly of pre-replicative complex (pre-RC) at origin DNA for replication licensing. Here we show that the N-terminal intrinsically disordered region (IDR) of the yeast Orc2 subunit is crucial for this process. Removing a segment (residues 176-200) from Orc2-IDR or mutating a key isoleucine (194) significantly inhibits replication initiation across the genome. These Orc2-IDR mutants are capable of assembling the ORC-Cdc6-Cdt1-Mcm2-7 intermediate, which exhibits impaired ATP hydrolysis and fails to be convered into the subsequent Mcm2-7-ORC complex and pre-RC. These defects can be partially rescued by the Orc2-IDR peptide. Moreover, the phosphorylation of this Orc2-IDR region by S cyclin-dependent kinase blocks its binding to Mcm2-7 complex, causing a defective pre-RC assembly. Our findings provide important insights into the multifaceted roles of ORC in supporting origin licensing during the G1 phase and its regulation to restrict origin firing within the S phase.

Polymers for Disrupting Protein-Protein Interactions: Where Are We and Where Should We Be?

Protein-protein interactions (PPIs) are central to the cellular signaling and regulatory networks that underlie many physiological and pathophysiological processes. It is challenging to target PPIs using traditional small molecule or peptide-based approaches due to the frequent lack of well-defined binding pockets at the large and flat PPI interfaces. Synthetic polymers offer an opportunity to circumvent these challenges by providing unparalleled flexibility in tuning their physiochemical properties to achieve the desired binding properties. In this review, we summarize the current state of the field pertaining to polymer-protein interactions in solution, highlighting various polyelectrolyte systems, their tunable parameters, and their characterization. We provide an outlook on how these architectures can be improved by incorporating sequence control, foldability, and machine learning to mimic proteins at every structural level. Advances in these directions will enable the design of more specific protein-binding polymers and provide an effective strategy for targeting dynamic proteins, such as intrinsically disordered proteins.

The microtubule regulator EFA-6 forms cortical foci dependent on its intrinsically disordered region and interactions with tubulins.

The EFA6 protein family, originally identified as Sec7 guanine nucleotide exchange factors, has also been found to regulate cortical microtubule (MT) dynamics. Here, we find that in the mature C. elegans epidermal epithelium, EFA-6 forms punctate foci in specific regions of the apical cortex, dependent on its intrinsically disordered region (IDR). The EFA-6 IDR can form biomolecular condensates in vitro. In genetic screens for mutants with altered GFP::EFA-6 localization, we identified a gain-of-function (gf) mutation in α-tubulin tba-1 that induces ectopic EFA-6 foci in multiple cell types. Lethality of tba-1(gf) is partially suppressed by loss of function in efa-6. The ability of TBA-1(gf) to trigger ectopic EFA-6 foci requires ß-tubulin TBB-2 and the chaperon EVL-20/Arl2. tba-1(gf)-induced EFA-6 foci display slower turnover, contain the MT-associated protein TAC-1/TACC, and require the EFA-6 MT elimination domain (MTED). Our results reveal functionally important crosstalk between cellular tubulins and cortical MT regulators in vivo.

Ordering the disordered.

In this Voices article, we introduce seven impressive young group leaders that presented their work at the recent Gordon Research Conference "Biophysics and biology of intrinsically disordered proteins" in Les Diablerets, Switzerland. We asked them to tell us more about their careers and their fascinating research on proteins that do not adopt a single-folded structure.

Intrinsically disordered sequences can tune fungal growth and the cell cycle for specific temperatures.

Temperature can impact every reaction essential to a cell. For organisms that cannot regulate their own temperature, adapting to temperatures that fluctuate unpredictably and on variable timescales is a major challenge. Extremes in the magnitude and frequency of temperature changes are increasing across the planet, raising questions as to how the biosphere will respond. To examine mechanisms of adaptation to temperature, we collected wild isolates from different climates of the fungus Ashbya gossypii, which has a compact genome of only ∼4,600 genes. We found control of the nuclear division cycle and polarized morphogenesis, both critical processes for fungal growth, were temperature sensitive and varied among the isolates. The phenotypes were associated with naturally varying sequences within the glutamine-rich region (QRR) IDR of an RNA-binding protein called Whi3. This protein regulates both nuclear division and polarized growth via its ability to form biomolecular condensates. In cells and in cell-free reconstitution assays, we found that temperature tunes the properties of Whi3-based condensates. Exchanging Whi3 sequences between isolates was sufficient to rescue temperature-sensitive phenotypes, and specifically, a heptad repeat sequence within the QRR confers temperature-sensitive behavior. Together, these data demonstrate that sequence variation in the size and composition of an IDR can promote cell adaptation to growth at specific temperature ranges. These data demonstrate the power of IDRs as tuning knobs for rapid adaptation to environmental fluctuations.