Analysis of molecular recognition features (MoRFs)

Several proteomic studies in the last decade revealed that many proteins are either completely disordered or possess long structurally flexible regions. Many such regions were shown to be of functional importance, often allowing a protein to interact with a large number of diverse partners. Parallel to these findings, during the last five years structural bioinformatics has produced an explosion of results regarding protein-protein interactions and their importance for cell signaling. We studied the occurrence of relatively short (10-70 residues), loosely structured protein regions within longer, largely disordered sequences that were characterized as bound to larger proteins. We call these regions molecular recognition features (MoRFs, also known as molecular recognition elements, MoREs). Interestingly, upon binding to their partner(s), MoRFs undergo disorder-to-order transitions. Thus, in our interpretation, MoRFs represent a class of disordered region that exhibits molecular recognition and binding functions. This work extends previous research showing the importance of flexibility and disorder for molecular recognition. We describe the development of a database of MoRFs derived from the RCSB Protein Data Bank and present preliminary results of bioinformatics analyses of these sequences. Based on the structure adopted upon binding, at least three basic types of MoRFs are found: α-MoRFs, β-MoRFs, and ι-MoRFs, which form α-helices, β-strands, and irregular secondary structure when bound, respectively. Our data suggest that functionally significant residual structure can exist in MoRF regions prior to the actual binding event. The contribution of intrinsic protein disorder to the nature and function of MoRFs has also been addressed. The results of this study will advance the understanding of protein-protein interactions and help towards the future development of useful protein-protein binding site predictors.     

Intrinsic disorder mediates hepatitis C virus core–host cell protein interactions

Viral proteins bind to numerous cellular and viral proteins throughout the infection cycle. However, the mechanisms by which viral proteins interact with such large numbers of factors remain unknown. Cellular proteins that interact with multiple, distinct partners often do so through short sequences known as molecular recognition features (MoRFs) embedded within intrinsically disordered regions (IDRs). In this study, we report the first evidence that MoRFs in viral proteins play a similar role in targeting the host cell. Using a combination of evolutionary modeling, protein–protein interaction analyses and forward genetic screening, we systematically investigated two computationally predicted MoRFs within the N‐terminal IDR of the hepatitis C virus (HCV) Core protein. Sequence analysis of the MoRFs showed their conservation across all HCV genotypes and the canine and equine Hepaciviruses. Phylogenetic modeling indicated that the Core MoRFs are under stronger purifying selection than the surrounding sequence, suggesting that these modules have a biological function. Using the yeast two‐hybrid assay, we identified three cellular binding partners for each HCV Core MoRF, including two previously characterized cellular targets of HCV Core (DDX3X and NPM1). Random and site‐directed mutagenesis demonstrated that the predicted MoRF regions were required for binding to the cellular proteins, but that different residues within each MoRF were critical for binding to different partners. This study demonstrated that viruses may use intrinsic disorder to target multiple cellular proteins with the same amino acid sequence and provides a framework for characterizing the binding partners of other disordered regions in viral and cellular proteomes.     

Molecular Recognition Features in Zika Virus Proteome

Viruses have compact genomes that encode limited number of proteins in comparison to other biological entities. Interestingly, viral proteins have shown natural abundance of either completely disordered proteins that are recognized as intrinsically disorder proteins (IDPs) or partially disordered segments known as intrinsically disordered protein regions (IDPRs). IDPRs are involved in interactions with multiple binding partners to accomplish signaling, regulation, and control functions in cells. Tuning of IDPs and IDPRs are mediated through post-translational modification and alternative splicing. Often, the interactions of IDPRs with their binding protein partner(s) lead to transition from the state of disorder to ordered form. Such interaction-prone protein IDPRs are identified as molecular recognition features (MoRFs). Molecular recognition is an important initial step for the biomolecular interactions and their functional proceedings. Although previous studies have established occurrence of the IDPRs in Zika virus proteome, which provide the functional diversity and structural plasticity to viral proteins, the MoRF analysis has not been performed as of yet. Many computational methods have been developed for the identification of the MoRFs in protein sequences including ANCHOR, MoRFpred, DISOPRED3, and MoRFchibi_web server. In the current study, we have investigated the presence of MoRF regions in structural and non-structural proteins of Zika virus using an aforementioned set of computational techniques. Furthermore, we have experimentally validated the intrinsic disorderness of NS2B cofactor region of NS2B–NS3 protease. NS2B has one of the longest MoRF regions in Zika virus proteome. In future, this study may provide valuable information while investigating the virus host protein interaction networks.     

SPOT-Disorder2: Improved Protein Intrinsic Disorder Prediction by Ensembled Deep Learning

Intrinsically disordered or unstructured proteins (or regions in proteins) have been found to be important in a wide range of biological functions and implicated in many diseases. Due to the high cost and low efficiency of experimental determination of intrinsic disorder and the exponential increase of unannotated protein sequences, developing complementary computational prediction methods has been an active area of research for several decades. Here, we employed an ensemble of deep Squeeze-and-Excitation residual inception and long short-term memory (LSTM) networks for predicting protein intrinsic disorder with input from evolutionary information and predicted one-dimensional structural properties. The method, called SPOT-Disorder2, offers substantial and consistent improvement not only over our previous technique based on LSTM networks alone, but also over other state-of-the-art techniques in three independent tests with different ratios of disordered to ordered amino acid residues, and for sequences with either rich or limited evolutionary information. More importantly, semi-disordered regions predicted in SPOT-Disorder2 are more accurate in identifying molecular recognition features (MoRFs) than methods directly designed for MoRFs prediction. SPOT-Disorder2 is available as a web server and as a standalone program at https://sparks-lab.org/server/spot-disorder2/.     

Abundance and functional roles of intrinsic disorder in the antimicrobial peptides of the NK-lysin family

NK-lysins are antimicrobial peptides (AMPs) that participate in the innate immune response and also have several pivotal roles in various biological processes. Such multifunctionality is commonly found among intrinsically disordered proteins. However, NK-lysins have never been systematically analyzed for intrinsic disorder. To fill this gap, the amino acid sequences of NK-lysins from various species were collected from UniProt and used for the comprehensive computational analysis to evaluate the propensity of these proteins for intrinsic disorder and to investigate the potential roles of disordered regions in NK-lysin functions. We analyzed abundance and peculiarities of intrinsic disorder distribution in all-known NK-lysins and showed that many NK-lysins are expected to have substantial levels of intrinsic disorder. Curiously, high level of intrinsic disorder was also found even in two proteins with known 3D-strucutres (NK-lysin from pig and human granulysin). Many of the identified disordered regions can be involved in protein–protein interactions. In fact, NK-lysins are shown to contain three to eight molecular recognition features; i.e. short structure-prone segments which are located within the long disordered regions and have a potential to undergo a disorder-to-order transition upon binding to a partner. Furthermore, these disordered regions are expected to have several sites of various posttranslational modifications. Our study shows that NK-lysins, which are AMPs with a set of prominent roles in the innate immune response, are expected to abundantly possess intrinsically disordered regions that might be related to multifunctionality of these proteins in the signal transduction pathways controlling the host response to pathogenic agents.     

OPAL+: Length‐Specific MoRF Prediction in Intrinsically Disordered Protein Sequences

Intrinsically disordered proteins (IDPs) contain long unstructured regions, which play an important role in their function. These intrinsically disordered regions (IDRs) participate in binding events through regions called molecular recognition features (MoRFs). Computational prediction of MoRFs helps identify the potentially functional regions in IDRs. In this study, OPAL+, a novel MoRF predictor, is presented. OPAL+ uses separate models to predict MoRFs of varying lengths along with incorporating the hidden Markov model (HMM) profiles and physicochemical properties of MoRFs and their flanking regions. Together, these features help OPAL+ achieve a marginal performance improvement of 0.4–0.7% over its predecessor for diverse MoRF test sets. This performance improvement comes at the expense of increased run time as a result of the requirement of HMM profiles. OPAL+ is available for download at https://github.com/roneshsharma/OPAL-plus/wiki/OPAL-plus-Download .     

Intrinsically disordered domains: Sequence ➔ disorder ➔ function relationships

Disordered domains are long regions of intrinsic disorder that ideally have conserved sequences, conserved disorder, and conserved functions. These domains were first noticed in protein–protein interactions that are distinct from the interactions between two structured domains and the interactions between structured domains and linear motifs or molecular recognition features (MoRFs). So far, disordered domains have not been systematically characterized. Here, we present a bioinformatics investigation of the sequence–disorder–function relationships for a set of probable disordered domains (PDDs) identified from the Pfam database. All the Pfam seed proteins from those domains with at least one PDD sequence were collected. Most often, if a set contains one PDD sequence, then all members of the set are PDDs or nearly so. However, many seed sets have sequence collections that exhibit diverse proportions of predicted disorder and structure, thus giving the completely unexpected result that conserved sequences can vary substantially in predicted disorder and structure. In addition to the induction of structure by binding to protein partners, disordered domains are also induced to form structure by disulfide bond formation, by ion binding, and by complex formation with RNA or DNA. The two new findings, (a) that conserved sequences can vary substantially in their predicted disorder content and (b) that homologues from a single domain can evolve from structure to disorder (or vice versa), enrich our understanding of the sequence ? disorder ensemble ? function paradigm.     

Comparisons of Allergenic and Metazoan Parasite Proteins: Allergy the Price of Immunity

Allergic reactions can be considered as maladaptive IgE immune responses towards environmental antigens. Intriguingly, these mechanisms are observed to be very similar to those implicated in the acquisition of an important degree of immunity against metazoan parasites (helminths and arthropods) in mammalian hosts. Based on the hypothesis that IgE-mediated immune responses evolved in mammals to provide extra protection against metazoan parasites rather than to cause allergy, we predict that the environmental allergens will share key properties with the metazoan parasite antigens that are specifically targeted by IgE in infected human populations. We seek to test this prediction by examining if significant similarity exists between molecular features of allergens and helminth proteins that induce an IgE response in the human host. By employing various computational approaches, 2712 unique protein molecules that are known IgE antigens were searched against a dataset of proteins from helminths and parasitic arthropods, resulting in a comprehensive list of 2445 parasite proteins that show significant similarity through sequence and structure with allergenic proteins. Nearly half of these parasite proteins from 31 species fall within the 10 most abundant allergenic protein domain families (EF-hand, Tropomyosin, CAP, Profilin, Lipocalin, Trypsin-like serine protease, Cupin, BetV1, Expansin and Prolamin). We identified epitopic-like regions in 206 parasite proteins and present the first example of a plant protein (BetV1) that is the commonest allergen in pollen in a worm, and confirming it as the target of IgE in schistosomiasis infected humans. The identification of significant similarity, inclusive of the epitopic regions, between allergens and helminth proteins against which IgE is an observed marker of protective immunity explains the ‘off-target’ effects of the IgE-mediated immune system in allergy. All these findings can impact the discovery and design of molecules used in immunotherapy of allergic conditions.     

Characterization of molecular recognition features, MoRFs, and their binding partners

Molecular Recognition Features (MoRFs) are short, interaction-prone segments of protein disorder that undergo disorder-to-order transitions upon specific binding, representing a specific class of intrinsically disordered regions that exhibit molecular recognition and binding functions. MoRFs are common in various proteomes and occupy a unique structural and functional niche in which function is a direct consequence of intrinsic disorder. Example MoRFs collected from the Protein Data Bank (PDB) have been divided into three subtypes according to their structures in the bound state: alpha-MoRFs form alpha-helices, beta-MoRFs form beta-strands, and iota-MoRFs form structures without a regular pattern of backbone hydrogen bonds. These example MoRFs were indicated to be intrinsically disordered in the absence of their binding partners by several criteria. In this study, we used several geometric and physiochemical criteria to examine the properties of 62 alpha-, 20 beta-, and 176 iota-MoRF complex structures. Interface residues were examined by calculating differences in accessible surface area between the complex and isolated monomers. The compositions and physiochemical properties of MoRF and MoRF partner interface residues were compared to the interface residues of homodimers, heterodimers, and antigen-antibody complexes. Our analysis indicates that there are significant differences in residue composition and several geometric and physicochemical properties that can be used to discriminate, with a high degree of accuracy, between various interfaces in protein interaction data sets. Implications of these findings for the development of MoRF-partner interaction predictors are discussed. In addition, structural changes upon MoRF-to-partner complex formation were examined for several illustrative examples.     

Intrinsic Disorder in the BK Channel and Its Interactome

The large-conductance Ca²⁺-activated K⁺ (BK) channel is broadly expressed in various mammalian cells and tissues such as neurons, skeletal and smooth muscles, exocrine cells, and sensory cells of the inner ear. Previous studies suggest that BK channels are promiscuous binders involved in a multitude of protein-protein interactions. To gain a better understanding of the potential mechanisms underlying BK interactions, we analyzed the abundance, distribution, and potential mechanisms of intrinsic disorder in 27 BK channel variants from mouse cochlea, 104 previously reported BK-associated proteins (BKAPS) from cytoplasmic and membrane/cytoskeletal regions, plus BK β- and γ-subunits. Disorder was evaluated using the MFDp algorithm, which is a consensus-based predictor that provides a strong and competitive predictive quality and PONDR, which can determine long intrinsically disordered regions (IDRs). Disorder-based binding sites or molecular recognition features (MoRFs) were found using MoRFpred and ANCHOR. BKAP functions were categorized based on Gene Ontology (GO) terms. The analyses revealed that the BK variants contain a number of IDRs. Intrinsic disorder is also common in BKAPs, of which ∼5% are completely disordered. However, intrinsic disorder is very differently distributed within BK and its partners. Approximately 65% of the disordered segments in BK channels are long (IDRs) (>50 residues), whereas >60% of the disordered segments in BKAPs are short IDRs that range in length from 4 to 30 residues. Both α and γ subunits showed various amounts of disorder as did hub proteins of the BK interactome. Our analyses suggest that intrinsic disorder is important for the function of BK and its BKAPs. Long IDRs in BK are engaged in protein-protein and protein-ligand interactions, contain multiple post-translational modification sites, and are subjected to alternative splicing. The disordered structure of BK and its BKAPs suggests one of the underlying mechanisms of their interaction.     

Exploring the binding diversity of intrinsically disordered proteins involved in one‐to‐many binding

Molecular recognition features (MoRFs) are intrinsically disordered protein regions that bind to partners via disorder‐to‐order transitions. In one‐to‐many binding, a single MoRF binds to two or more different partners individually. MoRF‐based one‐to‐many protein–protein interaction (PPI) examples were collected from the Protein Data Bank, yielding 23 MoRFs bound to 2–9 partners, with all pairs of same‐MoRF partners having less than 25% sequence identity. Of these, 8 MoRFs were bound to 2–9 partners having completely different folds, whereas 15 MoRFs were bound to 2–5 partners having the same folds but with low sequence identities. For both types of partner variation, backbone and side chain torsion angle rotations were used to bring about the conformational changes needed to enable close fits between a single MoRF and distinct partners. Alternative splicing events (ASEs) and posttranslational modifications (PTMs) were also found to contribute to distinct partner binding. Because ASEs and PTMs both commonly occur in disordered regions, and because both ASEs and PTMs are often tissue‐specific, these data suggest that MoRFs, ASEs, and PTMs may collaborate to alter PPI networks in different cell types. These data enlarge the set of carefully studied MoRFs that use inherent flexibility and that also use ASE‐based and/or PTM‐based surface modifications to enable the same disordered segment to selectively associate with two or more partners. The small number of residues involved in MoRFs and in their modifications by ASEs or PTMs may simplify the evolvability of signaling network diversity.     

Analysis of Molecular Recognition Features (MoRFs) in membrane proteins

Molecular Recognition Features (MoRFs) are defined as short, intrinsically disordered regions in proteins that undergo disorder-to-order transition upon binding to their partners. As their name suggests, they are implicated in molecular recognition, which serves as the initial step for protein–protein interactions. Membrane proteins constitute approximately 30% of fully sequenced proteomes and are responsible for a wide variety of cellular functions. The aim of the current study was to identify and analyze MoRFs in membrane proteins. Two datasets of MoRFs, transmembrane and peripheral membrane protein MoRFs, were constructed from the Protein Data Bank, and sequence, structural and functional analysis was performed. Characterization of our datasets revealed their unique compositional biases and membrane protein MoRFs were categorized depending on their secondary structure after the interaction with their partners. Moreover, the position of transmembrane protein MoRFs in relation with the protein's topology was determined. Further studies were focused on functional analyses of MoRF-containing proteins and MoRFs' partners, associating them with protein binding, regulation and cell signaling, indicating half of them as putative hubs in protein–protein interaction networks. In conclusion, we provide insights into the disorder-based protein–protein interactions involving membrane proteins.     

The intracellular distal tail of the Na+/H+ exchanger NHE1 is intrinsically disordered: implications for NHE1 trafficking

Intrinsic disorder is important for protein regulation, yet its role in regulation of ion transport proteins is essentially uninvestigated. The ubiquitous plasma membrane carrier protein Na(+)/H(+) Exchanger isoform 1 (NHE1) plays pivotal roles in cellular pH and volume homeostasis, and its dysfunction is implicated in several clinically important diseases. This study shows, for the first time for any carrier protein, that the distal part of the C-terminal intracellular tail (the cdt, residues V686-Q815) from human (h) NHE1 is intrinsically disordered. Further, we experimentally demonstrated the presence of a similar region of intrinsic disorder (ID) in NHE1 from the teleost fish Pleuronectes americanus (paNHE1), and bioinformatic analysis suggested ID to be conserved in the NHE1 family. The sequential variation in structure propensity as determined by NMR, but not the amplitude, was largely conserved between the h- and paNHE1cdt. This suggests that both proteins contain molecular recognition features (MoRFs), i.e., local, transiently formed structures within an ID region. The functional relevance of the most conserved MoRF was investigated by introducing a point mutation that significantly disrupted the putative binding feature. When this mutant NHE1 was expressed in full length NHE1 in AP1 cells, it exhibited impaired trafficking to the plasma membrane. This study demonstrated that the distal regulatory domain of NHE1 is intrinsically disordered yet contains conserved regions of transient structure. We suggest that normal NHE1 function depends on a protein recognition element within the ID region that may be linked to NHE1 trafficking via an acidic ER export motif.     

Intrinsic disorder of the extracellular matrix

The extracellular matrix is very well organized at the supramolecular and tissue levels and little is known on the potential role of intrinsic disorder in promoting its organization. We predicted the amount of disorder and identified disordered regions in the human extracellular proteome with established computational tools. The extracellular proteome is significantly enriched in proteins comprising more than 50% of disorder compared to the complete human proteome. The enrichment is mostly due to long disordered regions containing at least 100 consecutive disordered residues. The amount of intrinsic disorder is heterogeneous in the extracellular protein families, with the most disordered being collagens and the small integrin-binding ligand N-linked glycoproteins. Although most domains found in extracellular proteins are structured, the fibronectin III domains contain a variable amount of disordered residues (up to 92%). Binding sites for heparin and integrins are found in disordered sequences of extracellular proteins. Intrinsic disorder is evenly distributed in hubs and ends in the interaction network of extracellular proteins with their extracellular partners. In contrast, extracellular hubs are significantly enriched in disorder in the network of extracellular proteins with their extracellular, membrane and intracellular partners. Disorder could thus provide the structural plasticity required for the hubs to interact with membrane and intracellular proteins. Organization and assembly of the extracellular matrix, development of mineralized tissues and cell-matrix adhesion are the biological processes overrepresented in the most disordered extracellular proteins. Extracellular disorder is associated with binding to growth factors, glycosaminoglycans and integrins at the molecular level.     

Ordered Disorder of the Astrocytic Dystrophin-Associated Protein Complex in the Norm and Pathology

The abundance and potential functional roles of intrinsically disordered regions in aquaporin-4, Kir4.1, a dystrophin isoforms Dp71, α-1 syntrophin, and α-dystrobrevin; i.e., proteins constituting the functional core of the astrocytic dystrophin-associated protein complex (DAPC), are analyzed by a wealth of computational tools. The correlation between protein intrinsic disorder, single nucleotide polymorphisms (SNPs) and protein function is also studied together with the peculiarities of structural and functional conservation of these proteins. Our study revealed that the DAPC members are typical hybrid proteins that contain both ordered and intrinsically disordered regions. Both ordered and disordered regions are important for the stabilization of this complex. Many disordered binding regions of these five proteins are highly conserved among vertebrates. Conserved eukaryotic linear motifs and molecular recognition features found in the disordered regions of five protein constituting DAPC likely enhance protein-protein interactions that are required for the cellular functions of this complex. Curiously, the disorder-based binding regions are rarely affected by SNPs suggesting that these regions are crucial for the biological functions of their corresponding proteins.