HYPK: A marginally disordered protein sensitive to charge decoration.

Intrinsically disordered proteins (IDPs) that lie close to the empirical boundary separating IDPs and folded proteins in Uversky's charge-hydropathy plot may behave as "marginal IDPs" and sensitively switch conformation upon changes in environment (temperature, crowding, and charge screening), sequence, or both. In our search for such a marginal IDP, we selected Huntingtin-interacting protein K (HYPK) near that boundary as a candidate; PKIα, also near that boundary, has lower secondary structure propensity; and Crk1, just across the boundary on the folded side, has higher secondary structure propensity. We used a qualitative Förster resonance energy transfer-based assay together with circular dichroism to simultaneously probe global and local conformation. HYPK shows several unique features indicating marginality: a cooperative transition in end-to-end distance with temperature, like Crk1 and folded proteins, but unlike PKIα; enhanced secondary structure upon crowding, in contrast to Crk1 and PKIα; and a cross-over from salt-induced expansion to compaction at high temperature, likely due to a structure-to-disorder transition not seen in Crk1 and PKIα. We then tested HYPK's sensitivity to charge patterning by designing charge-flipped variants including two specific sequences with identical amino acid composition that markedly differ in their predicted size and response to salt. The experimentally observed trends, also including mutants of PKIα, verify the predictions from sequence charge decoration metrics. Marginal proteins like HYPK show features of both folded and disordered proteins that make them sensitive to physicochemical perturbations and structural control by charge patterning.

Functional in vitro diversity of an intrinsically disordered plant protein during freeze-thawing is encoded by its structural plasticity.

Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix-helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins.

Best practices for the manual curation of intrinsically disordered proteins in DisProt.

The DisProt database is a resource containing manually curated data on experimentally validated intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) from the literature. Developed in 2005, its primary goal was to collect structural and functional information into proteins that lack a fixed three-dimensional structure. Today, DisProt has evolved into a major repository that not only collects experimental data but also contributes to our understanding of the IDPs/IDRs roles in various biological processes, such as autophagy or the life cycle mechanisms in viruses or their involvement in diseases (such as cancer and neurodevelopmental disorders). DisProt offers detailed information on the structural states of IDPs/IDRs, including state transitions, interactions and their functions, all provided as curated annotations. One of the central activities of DisProt is the meticulous curation of experimental data from the literature. For this reason, to ensure that every expert and volunteer curator possesses the requisite knowledge for data evaluation, collection and integration, training courses and curation materials are available. However, biocuration guidelines concur on the importance of developing robust guidelines that not only provide critical information about data consistency but also ensure data acquisition.This guideline aims to provide both biocurators and external users with best practices for manually curating IDPs and IDRs in DisProt. It describes every step of the literature curation process and provides use cases of IDP curation within DisProt. Database URL: https://disprot.org/.

Rheostats, toggles, and neutrals, Oh my! A new framework for understanding how amino acid changes modulate protein function.

Advances in personalized medicine and protein engineering require accurately predicting outcomes of amino acid substitutions. Many algorithms correctly predict that evolutionarily-conserved positions show "toggle" substitution phenotypes, which is defined when a few substitutions at that position retain function. In contrast, predictions often fail for substitutions at the less-studied "rheostat" positions, which are defined when different amino acid substitutions at a position sample at least half of the possible functional range. This review describes efforts to understand the impact and significance of rheostat positions: (1) They have been observed in globular soluble, integral membrane, and intrinsically disordered proteins; within single proteins, their prevalence can be up to 40%. (2) Substitutions at rheostat positions can have biological consequences and ∼10% of substitutions gain function. (3) Although both rheostat and "neutral" (defined when all substitutions exhibit wild-type function) positions are nonconserved, the two classes have different evolutionary signatures. (4) Some rheostat positions have pleiotropic effects on function, simultaneously modulating multiple parameters (e.g., altering both affinity and allosteric coupling). (5) In structural studies, substitutions at rheostat positions appear to cause only local perturbations; the overall conformations appear unchanged. (6) Measured functional changes show promising correlations with predicted changes in protein dynamics; the emergent properties of predicted, dynamically coupled amino acid networks might explain some of the complex functional outcomes observed when substituting rheostat positions. Overall, rheostat positions provide unique opportunities for using single substitutions to tune protein function. Future studies of these positions will yield important insights into the protein sequence/function relationship.

Charge-Patterned Disordered Peptides Tune Intracellular Phase Separation in Bacteria.

Subcellular phase-separated compartments, known as biomolecular condensates, play an important role in the spatiotemporal organization of cells. To understand the sequence-determinants of phase separation in bacteria, we engineered protein-based condensates in Escherichia coli using electrostatic interactions as the main driving force. Minimal cationic disordered peptides were used to supercharge negative, neutral, and positive globular model proteins, enabling their phase separation with anionic biomacromolecules in the cell. The phase behavior was governed by the interaction strength between the cationic proteins and anionic biopolymers, in addition to the protein concentration. The interaction strength primarily depended on the overall net charge of the protein, but the distribution of charge between the globular and disordered domains also had an impact. Notably, the protein charge distribution between domains could tune mesoscale attributes such as the size, number, and subcellular localization of condensates within E. coli cells. The length and charge density of the disordered peptides had significant effects on protein expression levels, ultimately influencing the formation of condensates. Taken together, charge-patterned disordered peptides provide a platform for understanding the molecular grammar underlying phase separation in bacteria.

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The cellular response to mechanical stimuli depends largely on the structure of the cell itself and the abundance of intracellular cytomechanical proteins also plays a key role in the response to the stimulation of external mechanical signals. Liquid-liquid phase separation (LLPS) is the process by which proteins or protein-RNA complexes spontaneously separate and form two distinct "phases", ie, a low-concentration phase coexisting with a high-concentration phase. According to published findings, membrane-free organelles form and maintain their structures and regulate their internal biochemical activities through LLPS. LLPS, a novel mechanism for intracellular regulation of the biochemical reactions of biomacromolecules, plays a crucial role in modulating the responses of cytomechanical proteins. LLPS leads to the formation of highly concentrated liquid-phase condensates through multivalent interactions between biomacromolecules, thereby regulating a series of intracellular life activities. It has been reported that a variety of cytomechanical proteins respond to external mechanical signals through LLPS, which in turn affects biological behaviors such as cell growth, proliferation, spreading, migration, and apoptosis. Herein, we introduced the mechanisms of cytomechanics and LLPS. In addition, we presented the latest findings on cytomechanical protein phase separation, covering such issues as the regulation of focal adhesion maturation and mechanical signal transduction by LIM domain-containing protein 1 (LIMD1) phase separation, the regulation of intercellular tight junctions by zonula occludens (ZO) phase separation, and the regulation of cell proliferation and apoptosis by cytomechanical protein phase separation of the Hippo signaling pathway. The proposition of LLPS provides an explanation for the formation mechanism of intracellular membraneless organelles and supplies new approaches to understanding the biological functions of intracellular physiology or pathology. However, the molecular mechanisms by which LLPS drives focal adhesions and cell-edge dynamics are still not fully understood. It is not clear whether LLPS under in vitro conditions can occur under physiological conditions of organisms. There are still difficulties to be overcome in using LLPS to explain the interactions of multiple intracellular molecules. Researchers should pursue answers to these questions in the future.

The prefoldin-like protein AtURI exhibits characteristics of intrinsically disordered proteins.

The prefoldin-like protein UNCONVENTIONAL PREFOLDIN RPB5 INTERACTOR (URI) participates in diverse cellular functions, including protein homeostasis, transcription, translation, and signal transduction. Thus, URI is a highly versatile protein, although the molecular basis of this versatility remains unknown. In this work, we show that Arabidopsis thaliana (Arabidopsis) URI (AtURI) possesses a large intrinsically disordered region (IDR) spanning most of the C-terminal part of the protein, a feature conserved in yeast and human orthologs. Our findings reveal two key characteristics of disordered proteins in AtURI: promiscuity in interacting with partners and protein instability. We propose that these two features contribute to providing AtURI with functional versatility.

Decreasing the intrinsically disordered protein α-synuclein levels by targeting its structured mRNA with a ribonuclease-targeting chimera.

α-Synuclein is an important drug target for the treatment of Parkinson's disease (PD), but it is an intrinsically disordered protein lacking typical small-molecule binding pockets. In contrast, the encoding SNCA mRNA has regions of ordered structure in its 5' untranslated region (UTR). Here, we present an integrated approach to identify small molecules that bind this structured region and inhibit α-synuclein translation. A drug-like, RNA-focused compound collection was studied for binding to the 5' UTR of SNCA mRNA, affording Synucleozid-2.0, a drug-like small molecule that decreases α-synuclein levels by inhibiting ribosomes from assembling onto SNCA mRNA. This RNA-binding small molecule was converted into a ribonuclease-targeting chimera (RiboTAC) to degrade cellular SNCA mRNA. RNA-seq and proteomics studies demonstrated that the RiboTAC (Syn-RiboTAC) selectively degraded SNCA mRNA to reduce its protein levels, affording a fivefold enhancement of cytoprotective effects as compared to Synucleozid-2.0. As observed in many diseases, transcriptome-wide changes in RNA expression are observed in PD. Syn-RiboTAC also rescued the expression of ~50% of genes that were abnormally expressed in dopaminergic neurons differentiated from PD patient-derived iPSCs. These studies demonstrate that the druggability of the proteome can be expanded greatly by targeting the encoding mRNAs with both small molecule binders and RiboTAC degraders.

Conformational ensembles of the human intrinsically disordered proteome.

Intrinsically disordered proteins and regions (collectively, IDRs) are pervasive across proteomes in all kingdoms of life, help to shape biological functions and are involved in numerous diseases. IDRs populate a diverse set of transiently formed structures and defy conventional sequence-structure-function relationships1. Developments in protein science have made it possible to predict the three-dimensional structures of folded proteins at the proteome scale2. By contrast, there is a lack of knowledge about the conformational properties of IDRs, partly because the sequences of disordered proteins are poorly conserved and also because only a few of these proteins have been characterized experimentally. The inability to predict structural properties of IDRs across the proteome has limited our understanding of the functional roles of IDRs and how evolution shapes them. As a supplement to previous structural studies of individual IDRs3, we developed an efficient molecular model to generate conformational ensembles of IDRs and thereby to predict their conformational properties from sequences4,5. Here we use this model to simulate nearly all of the IDRs in the human proteome. Examining conformational ensembles of 28,058 IDRs, we show how chain compaction is correlated with cellular function and localization. We provide insights into how sequence features relate to chain compaction and, using a machine-learning model trained on our simulation data, show the conservation of conformational properties across orthologues. Our results recapitulate observations from previous studies of individual protein systems and exemplify how to link-at the proteome scale-conformational ensembles with cellular function and localization, amino acid sequence, evolutionary conservation and disease variants. Our freely available database of conformational properties will encourage further experimental investigation and enable the generation of hypotheses about the biological roles and evolution of IDRs.

How intrinsically disordered proteins order plant gene silencing.

Intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) possess low sequence complexity of amino acids and display non-globular tertiary structures. They can act as scaffolds, form regulatory hubs, or trigger biomolecular condensation to control diverse aspects of biology. Emerging evidence has recently implicated critical roles of IDPs and IDR-contained proteins in nuclear transcription and cytoplasmic post-transcriptional processes, among other molecular functions. We here summarize the concepts and organizing principles of IDPs. We then illustrate recent progress in understanding the roles of key IDPs in machineries that regulate transcriptional and post-transcriptional gene silencing (PTGS) in plants, aiming at highlighting new modes of action of IDPs in controlling biological processes.

Self-Assembling Polypeptides in Complex Coacervation.

ConspectusIntracellular compartmentalization plays a pivotal role in cellular function, with membrane-bound organelles and membrane-less biomolecular "condensates" playing key roles. These condensates, formed through liquid-liquid phase separation (LLPS), enable selective compartmentalization without the barrier of a lipid bilayer, thereby facilitating rapid formation and dissolution in response to stimuli. Intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs), which are often rich in charged and polar amino acid sequences, scaffold many condensates, often in conjunction with RNA.Comprehending the impact of IDP/IDR sequences on phase separation poses a challenge due to the extensive chemical diversity resulting from the myriad amino acids and post-translational modifications. To tackle this hurdle, one approach has been to investigate LLPS in simplified polypeptide systems, which offer a narrower scope within the chemical space for exploration. This strategy is supported by studies that have demonstrated how IDP function can largely be understood based on general chemical features, such as clusters or patterns of charged amino acids, rather than residue-level effects, and the ways in which these kinds of motifs give rise to an ensemble of conformations.Our laboratory has utilized complex coacervates assembled from oppositely charged polypeptides as a simplified material analogue to the complexity of liquid-liquid phase separated biological condensates. Complex coacervation is an associative LLPS that occurs due to the electrostatic complexation of oppositely charged macro-ions. This process is believed to be driven by the entropic gains resulting from the release of bound counterions and the reorganization of water upon complex formation. Apart from their direct applicability to IDPs, polypeptides also serve as excellent model polymers for investigating molecular interactions due to the wide range of available side-chain functionalities and the capacity to finely regulate their sequence, thus enabling precise control over interactions with guest molecules.Here, we discuss fundamental studies examining how charge patterning, hydrophobicity, chirality, and architecture affect the phase separation of polypeptide-based complex coacervates. These efforts have leveraged a combination of experimental and computational approaches that provide insight into molecular level interactions. We also examine how these parameters affect the ability of complex coacervates to incorporate globular proteins and viruses. These efforts couple directly with our fundamental studies into coacervate formation, as such "guest" molecules should not be considered as experiencing simple encapsulation and are instead active participants in the electrostatic assembly of coacervate materials. Interestingly, we observed trends in the incorporation of proteins and viruses into coacervates formed using different chain length polypeptides that are not well explained by simple electrostatic arguments and may be the result of more complex interactions between globular and polymeric species. Additionally, we describe experimental evidence supporting the potential for complex coacervates to improve the thermal stability of embedded biomolecules, such as viral vaccines.Ultimately, peptide-based coacervates have the potential to help unravel the physics behind biological condensates, while paving the way for innovative methods in compartmentalization, purification, and biomolecule stabilization. These advancements could have implications spanning medicine to biocatalysis.

Intrinsically disordered regions of the Msn2 transcription factor encode multiple functions using interwoven sequence grammars.

Intrinsically disordered regions (IDRs) are abundant in eukaryotic proteins, but their sequence-function relationship remains poorly understood. IDRs of transcription factors (TFs) can direct promoter selection and recruit coactivators, as shown for the budding yeast TF Msn2. To examine how IDRs encode both these functions, we compared genomic binding specificity, coactivator recruitment, and gene induction amongst a large set of designed Msn2-IDR mutants. We find that both functions depend on multiple regions across the > 600AA IDR. Yet, transcription activity was readily disrupted by mutations that showed no effect on the Msn2 binding specificity. Our data attribute this differential sensitivity to the integration of a relaxed, composition-based code directing binding specificity with a more stringent, motif-based code controlling the recruitment of coactivators and transcription activity. Therefore, Msn2 utilizes interwoven sequence grammars for encoding multiple functions, suggesting a new IDR design paradigm of potentially general use.

Disordered protein regions partner up the cBAF remodeler for a chromatin dance.

Intrinsically disordered protein regions form condensates and mediate interactions with factors that regulate gene activity. Patil et al.1 decode how such regions within the chromatin remodeler cBAF choreograph self-condensation and non-self interactions with transcriptional regulators, potentially impacting disease.

A disordered region controls cBAF activity via condensation and partner recruitment.

Intrinsically disordered regions (IDRs) represent a large percentage of overall nuclear protein content. The prevailing dogma is that IDRs engage in non-specific interactions because they are poorly constrained by evolutionary selection. Here, we demonstrate that condensate formation and heterotypic interactions are distinct and separable features of an IDR within the ARID1A/B subunits of the mSWI/SNF chromatin remodeler, cBAF, and establish distinct "sequence grammars" underlying each contribution. Condensation is driven by uniformly distributed tyrosine residues, and partner interactions are mediated by non-random blocks rich in alanine, glycine, and glutamine residues. These features concentrate a specific cBAF protein-protein interaction network and are essential for chromatin localization and activity. Importantly, human disease-associated perturbations in ARID1B IDR sequence grammars disrupt cBAF function in cells. Together, these data identify IDR contributions to chromatin remodeling and explain how phase separation provides a mechanism through which both genomic localization and functional partner recruitment are achieved.

Assessing the dynamics and macromolecular interactions of the intrinsically disordered protein YY1.

YY1 is a ubiquitously expressed, intrinsically disordered transcription factor involved in neural development. The oligomeric state of YY1 varies depending on the environment. These structural changes may alter its DNA binding ability and hence its transcriptional activity. Just as YY1's oligomeric state can impact its role in transcription, so does its interaction with other proteins such as FOXP2. The aim of this work is to study the structure and dynamics of YY1 so as to determine the influence of oligomerisation and associations with FOXP2 on its DNA binding mechanism. The results confirm that YY1 is primarily a disordered protein, but it does consist of certain specific structured regions. We observed that YY1 quaternary structure is a heterogenous mixture of oligomers, the overall size of which is dependent on ionic strength. Both YY1 oligomerisation and its dynamic behaviour are further subject to changes upon DNA binding, whereby increases in DNA concentration result in a decrease in the size of YY1 oligomers. YY1 and the FOXP2 forkhead domain were found to interact with each other both in isolation and in the presence of YY1-specific DNA. The heterogeneous, dynamic multimerisation of YY1 identified in this work is, therefore likely to be important for its ability to make heterologous associations with other proteins such as FOXP2. The interactions that YY1 makes with itself, FOXP2 and DNA form part of an intricate mechanism of transcriptional regulation by YY1, which is vital for appropriate neural development.

DeepDRP: Prediction of intrinsically disordered regions based on integrated view deep learning architecture from transformer-enhanced and protein information.

Intrinsic disorder in proteins, a widely distributed phenomenon in nature, is related to many crucial biological processes and various diseases. Traditional determination methods tend to be costly and labor-intensive, therefore it is desirable to seek an accurate identification method of intrinsically disordered proteins (IDPs). In this paper, we proposed a novel Deep learning model for Intrinsically Disordered Regions in Proteins named DeepDRP. DeepDRP employed an innovative TimeDistributed strategy and Bi-LSTM architecture to predict IDPs and is driven by integrated view features of PSSM, Energy-based encoding, AAindex, and transformer-enhanced embeddings including DR-BERT, OntoProtein, Prot-T5, and ESM-2. The comparison of different feature combinations indicates that the transformer-enhanced features contribute far more than traditional features to predict IDPs and ESM-2 accounts for a larger contribution in the pre-trained fusion vectors. The ablation test verified that the TimeDistributed strategy surely increased the model performance and is an efficient approach to the IDP prediction. Compared with eight state-of-the-art methods on the DISORDER723, S1, and DisProt832 datasets, the Matthews correlation coefficient of DeepDRP significantly outperformed competing methods by 4.90 % to 36.20 %, 11.80 % to 26.33 %, and 4.82 % to 13.55 %. In brief, DeepDRP is a reliable model for IDP prediction and is freely available at https://github.com/ZX-COLA/DeepDRP.

Periostin C-Terminal Is Intrinsically Disordered and Interacts with 143 Proteins in an In Vitro Epidermal Model of Atopic Dermatitis.

Human periostin is a 78-91 kDa matricellular protein implicated in extracellular matrix remodeling, tumor development, metastasis, and inflammatory diseases like atopic dermatitis, psoriasis, and asthma. The protein consists of six domains, including an N-terminal Cys-rich CROPT domain, four fasciclin-1 domains, and a C-terminal domain. The exons encoding the C-terminal domain may be alternatively spliced by shuffling four exons, generating ten variants of unknown function. Here, we investigate the structure and interactome of the full-length variant of the C-terminal domain with no exons spliced out. The structural analysis showed that the C-terminal domain lacked a tertiary structure and was intrinsically disordered. In addition, we show that the motif responsible for heparin-binding is in the conserved very C-terminal part of periostin. Pull-down confirmed three known interaction partners and identified an additional 140 proteins, among which nine previously have been implicated in atopic dermatitis. Based on our findings, we suggest that the C-terminal domain of periostin facilitates interactions between connective tissue components in concert with the four fasciclin domains.

Combined prediction and design reveals the target recognition mechanism of an intrinsically disordered protein interaction domain.

An increasing number of protein interaction domains and their targets are being found to be intrinsically disordered proteins (IDPs). The corresponding target recognition mechanisms are mostly elusive because of challenges in performing detailed structural analysis of highly dynamic IDP-IDP complexes. Here, we show that by combining recently developed computational approaches with experiments, the structure of the complex between the intrinsically disordered C-terminal domain (CTD) of protein 4.1G and its target IDP region in NuMA can be dissected at high resolution. First, we carry out systematic mutational scanning using dihydrofolate reductase-based protein complementarity analysis to identify essential interaction regions and key residues. The results are found to be highly consistent with an α/ß-type complex structure predicted by AlphaFold2 (AF2). We then design mutants based on the predicted structure using a deep learning protein sequence design method. The solved crystal structure of one mutant presents the same core structure as predicted by AF2. Further computational prediction and experimental assessment indicate that the well-defined core structure is conserved across complexes of 4.1G CTD with other potential targets. Thus, we reveal that an intrinsically disordered protein interaction domain uses an α/ß-type structure module formed through synergistic folding to recognize broad IDP targets. Moreover, we show that computational prediction and experiment can be jointly applied to segregate true IDP regions from the core structural domains of IDP-IDP complexes and to uncover the structure-dependent mechanisms of some otherwise elusive IDP-IDP interactions.

Chaotic aging: intrinsically disordered proteins in aging-related processes.

The development of aging is associated with the disruption of key cellular processes manifested as well-established hallmarks of aging. Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) have no stable tertiary structure that provide them a power to be configurable hubs in signaling cascades and regulate many processes, potentially including those related to aging. There is a need to clarify the roles of IDPs/IDRs in aging. The dataset of 1702 aging-related proteins was collected from established aging databases and experimental studies. There is a noticeable presence of IDPs/IDRs, accounting for about 36% of the aging-related dataset, which is however less than the disorder content of the whole human proteome (about 40%). A Gene Ontology analysis of the used here aging proteome reveals an abundance of IDPs/IDRs in one-third of aging-associated processes, especially in genome regulation. Signaling pathways associated with aging also contain IDPs/IDRs on different hierarchical levels, revealing the importance of "structure-function continuum" in aging. Protein-protein interaction network analysis showed that IDPs present in different clusters associated with different aging hallmarks. Protein cluster with IDPs enrichment has simultaneously high liquid-liquid phase separation (LLPS) probability, "nuclear" localization and DNA-associated functions, related to aging hallmarks: genomic instability, telomere attrition, epigenetic alterations, and stem cells exhaustion. Intrinsic disorder, LLPS, and aggregation propensity should be considered as features that could be markers of pathogenic proteins. Overall, our analyses indicate that IDPs/IDRs play significant roles in aging-associated processes, particularly in the regulation of DNA functioning. IDP aggregation, which can lead to loss of function and toxicity, could be critically harmful to the cell. A structure-based analysis of aging and the identification of proteins that are particularly susceptible to disturbances can enhance our understanding of the molecular mechanisms of aging and open up new avenues for slowing it down.

Exploring the evolution and function of Canoe's intrinsically disordered region in linking cell-cell junctions to the cytoskeleton during embryonic morphogenesis.

One central question for cell and developmental biologists is defining how epithelial cells can change shape and move during embryonic development without tearing tissues apart. This requires robust yet dynamic connections of cells to one another, via the cell-cell adherens junction, and of junctions to the actin and myosin cytoskeleton, which generates force. The last decade revealed that these connections involve a multivalent network of proteins, rather than a simple linear pathway. We focus on Drosophila Canoe, homolog of mammalian Afadin, as a model for defining the underlying mechanisms. Canoe and Afadin are complex, multidomain proteins that share multiple domains with defined and undefined binding partners. Both also share a long carboxy-terminal intrinsically disordered region (IDR), whose function is less well defined. IDRs are found in many proteins assembled into large multiprotein complexes. We have combined bioinformatic analysis and the use of a series of canoe mutants with early stop codons to explore the evolution and function of the IDR. Our bioinformatic analysis reveals that the IDRs of Canoe and Afadin differ dramatically in sequence and sequence properties. When we looked over shorter evolutionary time scales, we identified multiple conserved motifs. Some of these are predicted by AlphaFold to be alpha-helical, and two correspond to known protein interaction sites for alpha-catenin and F-actin. We next identified the lesions in a series of eighteen canoe mutants, which have early stop codons across the entire protein coding sequence. Analysis of their phenotypes are consistent with the idea that the IDR, including the conserved motifs in the IDR, are critical for protein function. These data provide the foundation for further analysis of IDR function.