[Human transcription factors as typical examples of those proteins with intrinsically disordered regions].
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Amino Acid Sequence↗
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Results for “Intrinsically Disordered Proteins”
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Rabies Virus Phosphoprotein Exhibits Thermoresponsive Phase Separation with a Lower Critical Solution Temperature.
Rabies virus (RABV) generates membrane-less liquid organelles (Negri bodies) in the cytoplasm of its host cell, where genome transcription and replication and nucleocapsid assembly take place, but the mechanisms of their assembly and maturation remain to be explained. An essential component of the viral RNA synthesizing machine, the phosphoprotein (P), acts as a scaffold protein for the assembly of these condensates. This intrinsically disordered protein forms star-shaped dimers with N-terminal negatively charged flexible arms and C-terminal globular domains exhibiting a large dipole moment. Our study shows that in vitro self-association of RABV P drives a complex thermoresponsive phase separation with a lower critical solution temperature. Protein dimers assemble already below the saturation concentration, and condensation is driven by attractive conformation-specific interactions leading to reentrant liquid phase separation over a narrow range of salt concentration. We propose a minimal molecular model in which P can adopt three limit conformational states and the disordered N-terminal arms control the interactions between giant dipoles that is consistent with our observations.
Rabies virus↗
Conservation of intrinsic disorder in protein domains and families: II. functions of conserved disorder.
Regions of conserved disorder prediction (CDP) were found in protein domains from all available InterPro member databases, although with varying frequency. These CDP regions were found in proteins from all kingdoms of life, including viruses. However, eukaryotes had 1 order of magnitude more proteins containing long disordered regions than did archaea and bacteria. Sequence conservation in CDP regions varied, but was on average slightly lower than in regions of conserved order. In some cases, disordered regions evolve faster than ordered regions, in others they evolve slower, and in the rest they evolve at roughly the same rate. A variety of functions were found to be associated with domains containing conserved disorder. The most common were DNA/RNA binding, and protein binding. Many ribosomal proteins also were found to contain conserved disordered regions. Other functions identified included membrane translocation and amino acid storage for germination. Due to limitations of current knowledge as well as the methodology used for this work, it was not determined whether these functions were directly associated with the predicted disordered region. However, the functions associated with conserved disorder in this work are in agreement with the functions found in other studies to correlate to disordered regions. We have established that intrinsic disorder may be more common in bacterial and archaeal proteins than previously thought, but this disorder is likely to be used for different purposes than in eukaryotic proteins, as well as occurring in shorter stretches of protein. Regions of predicted disorder were found to be conserved within a large number of protein families and domains. Although many think of such conserved domains as being ordered, in fact a significant number of them contain regions of disorder that are likely to be crucial to their functions.
Amino Acid Sequence↗
FlgM gains structure in living cells.
Intrinsically disordered proteins such as FlgM play important roles in biology, but little is known about their structure in cells. We use NMR to show that FlgM gains structure inside living Escherichia coli cells and under physiologically relevant conditions in vitro, i.e., in solutions containing high concentrations (>/=400 g/liter) of glucose, BSA, or ovalbumin. Structure formation represents solute-induced changes in the equilibrium between the structured and disordered forms of FlgM. The results provide insight into how the environment of intrinsically disordered proteins could dictate their structure and, in turn, emphasize the relevance of studying proteins in living cells and in vitro under physiologically realistic conditions.
Bacterial Proteins↗
Control of intrinsically disordered stathmin by multisite phosphorylation.
Stathmin is an intrinsically disordered protein implicated in the regulation of microtubule dynamics and in the development of cancer. The microtubule destabilizing activity of stathmin is down-regulated by phosphorylation of four serine residues, Ser16, Ser25, Ser38, and Ser63. Here we have used calorimetric and spectroscopic methods, including nuclear magnetic resonance to analyze the properties of seven stathmin phosphoisoforms to bind tubulin and inhibit microtubule formation. We found that stathmin phosphorylation results in a substantial loss in hydration entropy upon tubulin-stathmin complex formation. Remarkably, a linear correlation between the free energy change of complex formation and the microtubule inhibition activities of stathmin phosphoisoforms was observed. This finding provides a biophysical basis for understanding the mechanism by which local stathmin activity gradients important for promoting localized microtubule growth are established. We further found that phosphorylation of Ser16 and Ser63 disrupts the formation of a tubulin-interacting beta-hairpin and a helical segment, respectively, explaining the dominant role of these residues in regulating cell cycle progression. The insight into the tubulin-stathmin interaction offers a molecular basis for understanding the nature and the factors that control intrinsically disordered protein systems in general.
Circular Dichroism↗
Intrinsic disorder and protein function.
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Apoproteins↗
Drug discovery and p53.
In the past two decades, the identification of commonly mutated oncogenes and tumour suppressor genes has driven an unprecedented growth in our understanding of the genetic basis of human cancer. Although oncogenes can clearly serve as classically defined drug targets whose inactivation by small molecules could place a brake on cancer cell proliferation, the restoration of mutated tumour suppressor gene activity by small molecules might appear on the surface to be unrealistic. However, there is a growing realization that many eukaryotic regulatory proteins are partially unfolded and such intrinsically disordered proteins acquire a folded structure after binding to their biological target. Molecular characterization of the p53 protein has shown that its conformational flexibility and intrinsic thermodynamic instability provide a foundation from which its conformation can be quickly post-translationally modified.
Allosteric Regulation↗
Insights into protein structure and function from disorder-complexity space.
Intrinsically disordered proteins have a wide variety of important functional roles. However, the relationship between sequence and function in these proteins is significantly different than that for well-folded proteins. In a previous work, we showed that the propensity to be disordered can be recognized based on sequence composition alone. Here that analysis is furthered by examining the relationship of disorder propensity to sequence complexity, where the metrics for these two properties depend only on composition. The distributions of 40 amino acid peptides from both ordered and disordered proteins are graphed in this disorder-complexity space. An analysis of Swiss-Prot shows that most peptides have high complexity and relatively low disorder. However, there are also an appreciable number of low complexity-high disorder peptides in the database. In contrast, there are no low complexity-low disorder peptides. A similar analysis for peptides in the PDB reveals a much narrower distribution, with few peptides of low complexity and high disorder. In this case, the bounds of the disorder-complexity distribution are well defined and might be used to evaluate the likelihood that a peptide can be crystallized with current methods. The disorder-complexity distributions of individual proteins and sets of proteins grouped by function are also examined. Among individual proteins, there is an enormous variety of distributions that in some cases can be rationalized with regard to function. Groups of functionally related proteins are found to have distributions that are similar within each group but show notable differences between groups. Finally, a pattern matching algorithm is used to search for proteins with particular disorder-complexity distributions. The results suggest that this approach might be used to identify relationships between otherwise dissimilar proteins.
Algorithms↗
Intrinsic disorder in transcription factors.
Intrinsic disorder (ID) is highly abundant in eukaryotes, which reflect the greater need for disorder-associated signaling and transcriptional regulation in nucleated cells. Although several well-characterized examples of intrinsically disordered proteins in transcriptional regulation have been reported, no systematic analysis has been reported so far. To test for the general prevalence of intrinsic disorder in transcriptional regulation, we used the predictor of natural disorder regions (PONDR) to analyze the abundance of intrinsic disorder in three transcription factor datasets and two control sets. This analysis revealed that from 94.13 to 82.63% of transcription factors possess extended regions of intrinsic disorder, relative to 54.51 and 18.64% of the proteins in two control datasets, which indicates the significant prevalence of intrinsic disorder in transcription factors. This propensity of transcription factors to intrinsic disorder was confirmed by cumulative distribution function analysis and charge-hydropathy plots. The amino acid composition analysis showed that all three transcription factor datasets were substantially depleted in order-promoting residues and significantly enriched in disorder-promoting residues. Our analysis of the distribution of disorder within the transcription factor datasets revealed that (a) the AT-hooks and basic regions of transcription factor DNA-binding domains are highly disordered; (b) the degree of disorder in transcription factor activation regions is much higher than that in DNA-binding domains; (c) the degree of disorder is significantly higher in eukaryotic transcription factors than in prokaryotic transcription factors; and (d) the level of alpha-MoRF (molecular recognition feature) prediction is much higher in transcription factors. Overall, our data reflected the fact that eukaryotes with well-developed gene transcription machinery require transcription factor flexibility to be more efficient.
Amino Acid Sequence↗
Structural investigation of disordered stress proteins. Comparison of full-length dehydrins with isolated peptides of their conserved segments.
Dehydrins constitute a class of intrinsically disordered proteins that are expressed under conditions of water-related stress. Characteristic of the dehydrins are some highly conserved stretches of seven to 17 residues that are repetitively scattered in their sequences, the K-, S-, Y-, and Lys-rich segments. In this study, we investigate the putative role of these segments in promoting structure. The analysis is based on comparative analysis of four full-length dehydrins from Arabidopsis (Arabidopsis thaliana; Cor47, Lti29, Lti30, and Rab18) and isolated peptide mimics of the K-, Y-, and Lys-rich segments. In physiological buffer, the circular dichroism spectra of the full-length dehydrins reveal overall disordered structures with a variable content of poly-Pro helices, a type of elongated secondary structure relying on bridging water molecules. Similar disordered structures are observed for the isolated peptides of the conserved segments. Interestingly, neither the full-length dehydrins nor their conserved segments are able to adopt specific structure in response to altered temperature, one of the factors that regulate their expression in vivo. There is also no structural response to the addition of metal ions, increased protein concentration, or the protein-stabilizing salt Na(2)SO(4). Taken together, these observations indicate that the dehydrins are not in equilibrium with high-energy folded structures. The result suggests that the dehydrins are highly evolved proteins, selected to maintain high configurational flexibility and to resist unspecific collapse and aggregation. The role of the conserved segments is thus not to promote tertiary structure, but to exert their biological function more locally upon interaction with specific biological targets, for example, by acting as beads on a string for specific recognition, interaction with membranes, or intermolecular scaffolding. In this perspective, it is notable that the Lys-rich segment in Cor47 and Lti29 shows sequence similarity with the animal chaperone HSP90.
Amino Acid Sequence↗
Super-enhancer trapping by the nuclear pore via intrinsically disordered regions of proteins in squamous cell carcinoma cells.
Master transcription factors such as TP63 establish super-enhancers (SEs) to drive core transcriptional networks in cancer cells, yet the spatiotemporal regulation of SEs within the nucleus remains unknown. The nuclear pore complex (NPC) may tether SEs to the nuclear pore where RNA export rates are maximal. Here, we report that NUP153, a component of the NPC, anchors SEs to the NPC and enhances TP63 expression by maximizing mRNA export. This anchoring is mediated through protein-protein interaction between the intrinsically disordered regions (IDRs) of NUP153 and the coactivator BRD4. Silencing of NUP153 excludes SEs from the nuclear periphery, decreases TP63 expression, impairs cellular growth, and induces epidermal differentiation of squamous cell carcinoma. Overall, this work reveals the critical roles of NUP153 IDRs in the regulation of SE localization, thus providing insights into a new layer of gene regulation at the epigenomic and spatial level.
Humans↗
Coupling of folding and binding for unstructured proteins.
There are now numerous examples of proteins that are unstructured or only partially structured under physiological conditions and yet are nevertheless functional. Such proteins are especially prevalent in eukaryotes. In many cases, intrinsically disordered proteins adopt folded structures upon binding to their biological targets. Many new examples of coupled folding and binding events have been reported recently, providing new insights into mechanisms of molecular recognition.
Animals↗
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.
Algorithms↗
Intrinsic disorder in cell-signaling and cancer-associated proteins.
The number of intrinsically disordered proteins known to be involved in cell-signaling and regulation is growing rapidly. To test for a generalized involvement of intrinsic disorder in signaling and cancer, we applied a neural network predictor of natural disordered regions (PONDR VL-XT) to four protein datasets: human cancer-associated proteins (HCAP), signaling proteins (AfCS), eukaryotic proteins from SWISS-PROT (EU_SW) and non-homologous protein segments with well-defined (ordered) 3D structure (O_PDB_S25). PONDR VL-XT predicts >or=30 consecutive disordered residues for 79(+/-5)%, 66(+/-6)%, 47(+/-4)% and 13(+/-4)% of the proteins from HCAP, AfCS, EU_SW, and O_PDB_S25, respectively, indicating significantly more intrinsic disorder in cancer-associated and signaling proteins as compared to the two control sets. The disorder analysis was extended to 11 additional functionally diverse categories of human proteins from SWISS-PROT. The proteins involved in metabolism, biosynthesis, and degradation together with kinases, inhibitors, transport, G-protein coupled receptors, and membrane proteins are predicted to have at least twofold less disorder than regulatory, cancer-associated and cytoskeletal proteins. In contrast to 44.5% of the proteins from representative non-membrane categories, just 17.3% of the cancer-associated proteins had sequence alignments with structures in the Protein Data Bank covering at least 75% of their lengths. This relative lack of structural information correlated with the greater amount of predicted disorder in the HCAP dataset. A comparison of disorder predictions with the experimental structural data for a subset of the HCAP proteins indicated good agreement between prediction and observation. Our data suggest that intrinsically unstructured proteins play key roles in cell-signaling, regulation and cancer, where coupled folding and binding is a common mechanism.
Amino Acid Sequence↗
Protein flexibility and intrinsic disorder.
Comparisons were made among four categories of protein flexibility: (1) low-B-factor ordered regions, (2) high-B-factor ordered regions, (3) short disordered regions, and (4) long disordered regions. Amino acid compositions of the four categories were found to be significantly different from each other, with high-B-factor ordered and short disordered regions being the most similar pair. The high-B-factor (flexible) ordered regions are characterized by a higher average flexibility index, higher average hydrophilicity, higher average absolute net charge, and higher total charge than disordered regions. The low-B-factor regions are significantly enriched in hydrophobic residues and depleted in the total number of charged residues compared to the other three categories. We examined the predictability of the high-B-factor regions and developed a predictor that discriminates between regions of low and high B-factors. This predictor achieved an accuracy of 70% and a correlation of 0.43 with experimental data, outperforming the 64% accuracy and 0.32 correlation of predictors based solely on flexibility indices. To further clarify the differences between short disordered regions and ordered regions, a predictor of short disordered regions was developed. Its relatively high accuracy of 81% indicates considerable differences between ordered and disordered regions. The distinctive amino acid biases of high-B-factor ordered regions, short disordered regions, and long disordered regions indicate that the sequence determinants for these flexibility categories differ from one another, whereas the significantly-greater-than-chance predictability of these categories from sequence suggest that flexible ordered regions, short disorder, and long disorder are, to a significant degree, encoded at the primary structure level.
Amino Acid Sequence↗
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.
Algorithms↗
Sensitivity of secondary structure propensities to sequence differences between alpha- and gamma-synuclein: implications for fibrillation.
The synucleins are a family of intrinsically disordered proteins involved in various human diseases. alpha-Synuclein has been extensively characterized due to its role in Parkinson's disease where it forms intracellular aggregates, while gamma-synuclein is overexpressed in a majority of late-stage breast cancers. Despite fairly strong sequence similarity between the amyloid-forming regions of alpha- and gamma-synuclein, gamma-synuclein has only a weak propensity to form amyloid fibrils. We hypothesize that the different fibrillation tendencies of alpha- and gamma-synuclein may be related to differences in structural propensities. Here we have measured chemical shifts for gamma-synuclein and compared them to previously published shifts for alpha-synuclein. In order to facilitate direct comparison, we have implemented a simple new technique for re-referencing chemical shifts that we have found to be highly effective for both disordered and folded proteins. In addition, we have developed a new method that combines different chemical shifts into a single residue-specific secondary structure propensity (SSP) score. We observe significant differences between alpha- and gamma-synuclein secondary structure propensities. Most interestingly, gamma-synuclein has an increased alpha-helical propensity in the amyloid-forming region that is critical for alpha-synuclein fibrillation, suggesting that increased structural stability in this region may protect against gamma-synuclein aggregation. This comparison of residue-specific secondary structure propensities between intrinsically disordered homologs highlights the sensitivity of transient structure to sequence changes, which we suggest may have been exploited as an evolutionary mechanism for fast modulation of protein structure and, hence, function.
Amino Acid Sequence↗
The HPV16 E7 viral oncoprotein self-assembles into defined spherical oligomers.
Despite the fact that E7 is a major transforming oncoprotein in papillomavirus, its structure and precise molecular mechanism of action remain puzzling to date. E7 proteins share sequence homology and proteasome targeting properties of tumor suppressors with adenovirus E1A and SV40 T antigen, two other paradigmatic oncoproteins from DNA tumor viruses. High-risk HPV16 E7, a nonglobular dimer with some properties of intrinsically disordered proteins, is capable of undergoing pH-dependent conformational transitions that expose hydrophobic surfaces to the solvent. We found that treatment with a chelating agent produced a protein that can readily assemble into homogeneous spherical particles with an average molecular mass of 790 kDa and a diameter of 50 nm, as determined from dynamic light scattering and electron microscopy. The protein undergoes a substantial conformational transition from coil to beta-sheet structure, with concomitant consolidation of tertiary structure as judged by circular dichroism and fluorescence. The assembly process is very slow, in agreement with a substantial energy barrier caused by structural rearrangements. The resulting particles are highly stable, cooperatively folded, and capable of binding both Congo Red and thioflavin T, reporters of repetitive beta-sheet structures similar to those found in amyloids, although no fibrillar or insoluble material was observed under our experimental conditions.
Benzothiazoles↗