Abundance of intrinsic disorder in protein associated with cardiovascular disease

Evidence that many protein regions and even entire proteins lacking stable tertiary and/or secondary structure in solution (i.e., intrinsically disordered proteins) might be involved in protein-protein interactions, regulation, recognition, and signal transduction is rapidly accumulating. These signaling proteins play a crucial role in the development of several pathological conditions, including cancer. To test a hypothesis that intrinsic disorder is also abundant in cardiovascular disease (CVD), a data set of 487 CVD-related proteins was extracted from SWISS-PROT. CVD-related proteins are depleted in major order-promoting residues (Trp, Phe, Tyr, Ile, and Val) and enriched in some disorder-promoting residues (Arg, Gln, Ser, Pro, and Glu). The application of a neural network predictor of natural disordered regions (PONDR VL-XT) together with cumulative distribution function (CDF) analysis, charge-hydropathy plot (CH plot) analysis, and alpha-helical molecular recognition feature (alpha-MoRF) indicator revealed that CVD-related proteins are enriched in intrinsic disorder. In fact, the percentage of proteins with 30 or more consecutive residues predicted by PONDR VL-XT to be disordered was 57 +/- 4% for CVD-associated proteins. This value is close that described earlier for signaling proteins (66 +/- 6%) and is significantly larger than the content of intrinsic disorder in eukaryotic proteins from SWISS-PROT (47 +/- 4%) and in nonhomologous protein segments with a well-defined three-dimensional structure (13 +/- 4%). Furthermore, CDF and CH-plot analyses revealed that 120 and 36 CVD-related proteins, respectively, are wholly disordered. This high level of intrinsic disorder could be important for the function of CVD-related proteins and for the control and regulation of processes associated with cardiovascular disease. In agreement with this hypothesis, 198 alpha-MoRFs were predicted in 101 proteins from the CVD data set. A comparison of disorder predictions with the experimental structural and functional data for a subset of the CVD-associated proteins indicated good agreement between predictions and observations. Thus, our data suggest that intrinsically disordered proteins might play key roles in cardiovascular disease.     

The disordered amino-terminus of SIMPL interacts with members of the 70-kDa heat-shock protein family

The p65 coactivator SIMPL is a small protein that lacks any conserved domains of known function. To better understand regulation of SIMPL activity, we sought to identify novel SIMPL interacting proteins using mass spectrometry analysis of SIMPL containing complexes. Two members of the 70-kDa heat-shock protein family, Hsp70 and Hsc70, were identified as SIMPL binding proteins. Subsequent immunocomplexing assays confirmed this interaction and demonstrated that the amino-terminus of SIMPL is required for this interaction. Using a combination of amino acid composition analysis, PONDR VL-XT prediction, charge-hydropathy plots, and cumulative distribution functions, the amino-terminal region of both mouse and human SIMPL proteins was predicted to be intrinsically disordered. These data, taken together, suggest that Hsp70/Hsc70 bind the intrinsically disordered amino-terminal region of SIMPL to stabilize the protein and thereby regulate its activity. Understanding the regulation of SIMPL through its interaction with Hsp70/Hsc70 may serve as a novel means of modulating tumor necrosis factor alpha signaling.     

Natively Disordered Proteins

Proteins can exist in at least three forms: the ordered form (solid-like), the partially folded form (collapsed, molten globule-like or liquid-like) and the extended form (extended, random coil-like or gas-like). The protein trinity hypothesis has two components: (i) a given native protein can be in any one of the three forms, depending on the sequence and the environment; and (ii) function can arise from any one of the three forms or from transitions between them. In this study, bioinformatics and data mining were used to investigate intrinsic disorder in proteins and develop neural network-based predictors of natural disordered regions (PONDR) that can discriminate between ordered and disordered residues with up to 84% accuracy. Predictions of intrinsic disorder indicate that the three kingdoms follow the disorder ranking eubacteria < archaebacteria < eukaryotes, with approximately half of eukaryotic proteins predicted to contain substantial regions of intrinsic disorder. Many of the known disordered regions are involved in signalling, regulation or control. Involvement of highly flexible or disordered regions in signalling is logical: a flexible sensor more readily undergoes conformational change in response to environmental perturbations than does a rigid one. Thus, the increased disorder in the eukaryotes is likely the direct result of an increased need for signalling and regulation in nucleated organisms. PONDR can also be used to detect molecular recognition elements that are disordered in the unbound state and become structured when bound to a biologically meaningful partner. Application of disorder predictions to cell-signalling, cancer-associated and control protein databases supports the widespread occurrence of protein disorder in these processes.     

Long-range effects of tag sequence on marginally stabilized structure in HIV-1 p24 capsid protein monitored using NMR

N-terminal domain of HIV-1 p24 capsid protein is a globular fold composed of seven helices and two β-strands with a flexible structure including the α4–5 loop and both N- and C-terminal ends. However, the protein shows a high tendency (48%) for an intrinsically disordered structure based on the PONDR VL-XT prediction from the primary sequence. To assess the possibility of marginally stabilized structure under physiological conditions, the N-terminal domain of p24 was destabilized by the addition of an artificial flexible tag to either N- or C-terminal ends, and it was analyzed using T₁, T₂, hetero-nuclear NOE, and amide-proton exchange experiments. When the C-terminal tag (12 residues) was attached, the regions of the α3–4 loop and helix 6 as well as the α4–5 loop attained the flexible structures. Furthermore, in the protein containing the N-terminal tag (27 residues), helix 4 in addition to the above-mentioned area including α3–4 and α4–5 loops as well as helix 6 exhibited highly disordered structures. Thus, the long-range effects of the existence of tag sequence was observed in the stepwise manner of the appearance of disordered structures (step 1: α4–5 loop, step 2: α3–4 loop and helix 6, and step 3: helix 4). Furthermore, the disordered regions in tagged proteins were consistent with the PONDR VL-XT disordered prediction. The dynamic structure located in the middle part (α3–4 loop to helix 6) of the protein shown in this study may be related to the assembly of the viral particle.     

Optimizing long intrinsic disorder predictors with protein evolutionary information

Protein existing as an ensemble of structures, called intrinsically disordered, has been shown to be responsible for a wide variety of biological functions and to be common in nature. Here we focus on improving sequence-based predictions of long (>30 amino acid residues) regions lacking specific 3-D structure by means of four new neural-network-based Predictors Of Natural Disordered Regions (PONDRs): VL3, VL3H, VL3P, and VL3E. PONDR VL3 used several features from a previously introduced PONDR VL2, but benefitted from optimized predictor models and a slightly larger (152 vs. 145) set of disordered proteins that were cleaned of mislabeling errors found in the smaller set. PONDR VL3H utilized homologues of the disordered proteins in the training stage, while PONDR VL3P used attributes derived from sequence profiles obtained by PSI-BLAST searches. The measure of accuracy was the average between accuracies on disordered and ordered protein regions. By this measure, the 30-fold cross-validation accuracies of VL3, VL3H, and VL3P were, respectively, 83.6 +/- 1.4%, 85.3 +/- 1.4%, and 85.2 +/- 1.5%. By combining VL3H and VL3P, the resulting PONDR VL3E achieved an accuracy of 86.7 +/- 1.4%. This is a significant improvement over our previous PONDRs VLXT (71.6 +/- 1.3%) and VL2 (80.9 +/- 1.4%). The new disorder predictors with the corresponding datasets are freely accessible through the web server at http://www.ist.temple.edu/disprot.     

The roles of intrinsic disorder in orchestrating the Wnt-pathway

The canonical Wnt-pathway plays a number of crucial roles in the development of organism. Malfunctions of this pathway lead to various diseases including cancer. In the inactivated state, this pathway involves five proteins, Axin, CKI-α, GSK-3β, APC, and β-catenin. We analyzed these proteins by a number of computational tools, such as PONDR(r)VLXT, PONDR(r)VSL2, MoRF-II predictor and Hydrophobic Cluster Analysis (HCA) to show that each of the Wnt-pathway proteins contains several intrinsically disordered regions. Based on a comprehensive analysis of published data we conclude that these disordered regions facilitate protein-protein interactions, post-translational modifications, and signaling. The scaffold protein Axin and another large protein, APC, act as flexible concentrators in gathering together all other proteins involved in the Wnt-pathway, emphasizing the role of intrinsically disordered regions in orchestrating the complex protein-protein interactions. We further explore the intricate roles of highly disordered APC in regulation of β-catenin function. Intrinsically disordered APC helps the collection of β-catenin from cytoplasm, facilitates the b-catenin delivery to the binding sites on Axin, and controls the final detachment of β-catenin from Axin.     

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.     

Intrinsic disorder in S100 proteins

Although the members of the largest subfamily of the EF-hand proteins, S100 proteins, are evolutionarily young, their functional diversity is extremely broad, partly due to their ability to adapt to various targets. This feature is a hallmark of intrinsically disordered proteins (IDPs), but none of the S100 proteins are recognized as IDPs. S100 are predicted to be enriched in intrinsic disorder, with 62% of them being predicted to be disordered by at least one of the predictors: 31% are recognized as 'molten globules' and 15% are shown to be in extended disordered form. The disorder level of predicted disordered S100 regions is conserved compared to that of more structured regions. The central disordered stretch corresponds to the major part of pseudo EF-hand loop, helix II, hinge region, and an initial part of helix III. It contains about half of known sites of enzymatic post-translational modifications (PTMs), confirming that this region can be flexible in vivo. Most of the internal residues missing in tertiary structures belong to the hinge. Both hinge and pseudo EF-hand loop correspond to the local maxima of the PONDR? VSL2 score and are shown to be evolutionary hotspots, leading to gain of new functional properties. The action of PTMs is shown to be destabilizing, in contrast with the effect of metal-binding or S100 dimerization. Formation of the S100 heterodimers relies on the interplay between the structural rigidity of one of the S100 monomers and the flexibility of another monomer. The ordered regions dominate in the S100 homodimerization sites. Target-binding sites generally consist of distant regions, drastically differing in their disorder level. The disordered region comprising most of the hinge and the N-terminal half of helix III is virtually not involved into dimerization, being intended solely for target recognition. The structural flexibility of this region is essential for recognition of diverse target proteins. At least 86% of multiple interactions of S100 proteins with binding partners are attributed to the S100 proteins predicted to be disordered. Overall, the intrinsic disorder is inherent to many S100 proteins and is vital for activity and functional diversity of the family.     

Differences in the number of intrinsically disordered regions between yeast duplicated proteins, and their relationship with functional divergence

Background

Intrinsically disordered regions are enriched in short interaction motifs that play a critical role in many protein-protein interactions. Since new short interaction motifs may easily evolve, they have the potential to rapidly change protein interactions and cellular signaling. In this work we examined the dynamics of gain and loss of intrinsically disordered regions in duplicated proteins to inspect if changes after genome duplication can create functional divergence. For this purpose we used Saccharomyces cerevisiae and the outgroup species Lachancea kluyveri.

Principal Findings

We find that genes duplicated as part of a genome duplication (ohnologs) are significantly more intrinsically disordered than singletons (p<2.2^e-16, Wilcoxon), reflecting a preference for retaining intrinsically disordered proteins in duplicate. In addition, there have been marked changes in the extent of intrinsic disorder following duplication. A large number of duplicated genes have more intrinsic disorder than their L. kluyveri ortholog (29% for duplicates versus 25% for singletons) and an even greater number have less intrinsic disorder than the L. kluyveri ortholog (37% for duplicates versus 25% for singletons). Finally, we show that the number of physical interactions is significantly greater in the more intrinsically disordered ohnolog of a pair (p = 0.003, Wilcoxon).

Conclusion

This work shows that intrinsic disorder gain and loss in a protein is a mechanism by which a genome can also diverge and innovate. The higher number of interactors for proteins that have gained intrinsic disorder compared with their duplicates may reflect the acquisition of new interaction partners or new functional roles.     

Identification of Atg3 as an intrinsically disordered polypeptide yields insights into the molecular dynamics of autophagy-related proteins in yeast

The mechanism of autophagy relies on complex cell signaling and regulatory processes. Each cell contains many proteins that lack a rigid 3-dimensional structure under physiological conditions. These dynamic proteins, called intrinsically disordered proteins (IDPs) and protein regions (IDPRs), are predominantly involved in cell signaling and regulation. Yet, very little is known about their presence among proteins of the core autophagy machinery. In this work, we characterized the autophagy protein Atg3 from yeast and human along with 2 variants to show that Atg3 is an IDPRs-containing protein and that disorder/order predicted for these proteins from their amino acid sequence corresponds to their experimental characteristics. Based on this consensus, we applied the same prediction methods to all known Atg proteins from Saccharomyces cerevisiae. The data presented here provide an insight into the structural dynamics of each Atg protein. They also show that intrinsic disorder at various levels has to be taken into consideration for about half of the Atg proteins. This work should become a useful tool that will facilitate and encourage exploration of protein intrinsic disorder in autophagy.     

Understanding protein non-folding

This review describes the family of intrinsically disordered proteins, members of which fail to form rigid 3-D structures under physiological conditions, either along their entire lengths or only in localized regions. Instead, these intriguing proteins/regions exist as dynamic ensembles within which atom positions and backbone Ramachandran angles exhibit extreme temporal fluctuations without specific equilibrium values. Many of these intrinsically disordered proteins are known to carry out important biological functions which, in fact, depend on the absence of a specific 3-D structure. The existence of such proteins does not fit the prevailing structure–function paradigm, which states that a unique 3-D structure is a prerequisite to function. Thus, the protein structure–function paradigm has to be expanded to include intrinsically disordered proteins and alternative relationships among protein sequence, structure, and function. This shift in the paradigm represents a major breakthrough for biochemistry, biophysics and molecular biology, as it opens new levels of understanding with regard to the complex life of proteins. This review will try to answer the following questions: how were intrinsically disordered proteins discovered? Why don't these proteins fold? What is so special about intrinsic disorder? What are the functional advantages of disordered proteins/regions? What is the functional repertoire of these proteins? What are the relationships between intrinsically disordered proteins and human diseases?     

Identification of Atg3 as an intrinsically disordered polypeptide yields insights into the molecular dynamics of autophagy-related proteins in yeast

The mechanism of autophagy relies on complex cell signaling and regulatory processes. Each cell contains many proteins that lack a rigid 3-dimensional structure under physiological conditions. These dynamic proteins, called intrinsically disordered proteins (IDPs) and protein regions (IDPRs), are predominantly involved in cell signaling and regulation. Yet, very little is known about their presence among proteins of the core autophagy machinery. In this work, we characterized the autophagy protein Atg3 from yeast and human along with 2 variants to show that Atg3 is an IDPRs-containing protein and that disorder/order predicted for these proteins from their amino acid sequence corresponds to their experimental characteristics. Based on this consensus, we applied the same prediction methods to all known Atg proteins from Saccharomyces cerevisiae. The data presented here provide an insight into the structural dynamics of each Atg protein. They also show that intrinsic disorder at various levels has to be taken into consideration for about half of the Atg proteins. This work should become a useful tool that will facilitate and encourage exploration of protein intrinsic disorder in autophagy.     

Prevalent structural disorder in E. coli and S. cerevisiae proteomes

Intrinsically unstructured proteins, which exist without a well-defined 3D structure, carry out essential functions and occur with high frequency, as predicted for genomes. The generality of this phenomenon, however, is questioned by the uncertainty of what fraction of genomes actually encodes for expressed proteins. Here, we used two independent bioinformatic predictors, PONDR VSL1, and IUPred, to demonstrate that disorder prevails in the recently characterized proteomes and essential proteins of E. coli and S. cerevisiae, at levels exceeding that estimated from the genomes. The S. cerevisiae proteome contains three times as much disorder as that of E. coli, with 50-60% of proteins containing at least one long (>30 residues) disordered segment. This evolutionary advance can be explained by the observation that disorder is much higher in Gene Ontology categories related to regulatory, as opposed to metabolic, functions, and also in categories unique to yeast. Thus, protein disorder is a widespread and functionally important phenomenon, which needs to be characterized in full detail for understanding complex organisms at the molecular level.     

Intrinsic disorder in the Protein Data Bank

The Protein Data Bank (PDB) is the preeminent source of protein structural information. PDB contains over 32,500 experimentally determined 3-D structures solved using X-ray crystallography or nuclear magnetic resonance spectroscopy. Intrinsically disordered regions fail to form a fixed 3-D structure under physiological conditions. In this study, we compare the amino-acid sequences of proteins whose structures are determined by X-ray crystallography with the corresponding sequences from the Swiss-Prot database. The analyzed dataset includes 16,370 structures, which represent 18,101 PDB chains and 5,434 different proteins from 910 different organisms (2,793 eukaryotic, 2,109 bacterial, 288 viral, and 244 archaeal). In this dataset, on average, each Swiss-Prot protein is represented by 7 PDB chains with 76% of the crystallized regions being represented by more than one structure. Intriguingly, the complete sequences of only approximately 7% of proteins are observed in the corresponding PDB structures, and only approximately 25% of the total dataset have >95% of their lengths observed in the corresponding PDB structures. This suggests that the vast majority of PDB proteins is shorter than their corresponding Swiss-Prot sequences and/or contain numerous residues, which are not observed in maps of electron density. To determine the prevalence of disordered regions in PDB, the residues in the Swiss-Prot sequences were grouped into four general categories, "Observed" (which correspond to structured regions), "Not observed" (regions with missing electron density, potentially disordered), "Uncharacterized," and "Ambiguous," depending on their appearance in the corresponding PDB entries. This non-redundant set of residues can be viewed as a 'fragment' or empirical domain database that contains a set of experimentally determined structured regions or domains and a set of experimentally verified disordered regions or domains. We studied the propensities and properties of residues in these four categories and analyzed their relations to the predictions of disorder using several algorithms. "Non-observed," "Ambiguous," and "Uncharacterized" regions were shown to possess the amino acid compositional biases typical of intrinsically disordered proteins. The application of four different disorder predictors (PONDR(R) VL-XT, VL3-BA, VSL1P, and IUPred) revealed that the vast majority of residues in the "Observed" dataset are ordered, and that the "Not observed" regions are mostly disordered. The "Uncharacterized" regions possess some tendency toward order, whereas the predictions for the short "Ambiguous" regions are really ambiguous. Long "Ambiguous" regions (>70 amino acid residues) are mostly predicted to be ordered, suggesting that they are likely to be "wobbly" domains. Overall, we showed that completely ordered proteins are not highly abundant in PDB and many PDB sequences have disordered regions. In fact, in the analyzed dataset approximately 10% of the PDB proteins contain regions of consecutive missing or ambiguous residues longer than 30 amino-acids and approximately 40% of the proteins possess short regions (> or =10 and < 30 amino-acid long) of missing and ambiguous residues.     

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.     

More than just tails: intrinsic disorder in histone proteins

Many biologically active proteins are disordered as a whole, or contain long disordered regions. These intrinsically disordered proteins/regions are very common in nature, abundantly found in all organisms, where they carry out important biological functions. The functions of these proteins complement the functional repertoire of "normal" ordered proteins, and many protein functional classes are heavily dependent on intrinsic disorder. Among these disorder-centric functions are interactions with nucleic acids and protein complex assembly. In this study, we present the results of comprehensive bioinformatics analyses of the abundance and roles of intrinsic disorder in 2007 histones from 746 species. We show that all the members of the histone family are intrinsically disordered proteins. Furthermore, intrinsic disorder is not only abundant in histones, but is absolutely necessary for various histone functions, starting from heterodimerization to formation of higher order oligomers, to interactions with DNA and other proteins, and to posttranslational modifications.     

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.