Disorder-promoting Residues Research Articles

Amino acid pattern reveals multi-functionality of ORF3 protein from HEV.

The smallest open reading frame (ORF) encoded protein ORF3 of hepatitis E virus (HEV), recently, has been demonstrated to perform multiple functions besides accessory roles. ORF3 could act as a target for vaccine against HEV infections. The IDR (intrinsically disordered region); IDP (ID protein)/IDPR (ID protein region), plays critical role in various regulatory functions of viruses. The dark proteome of HEV-ORF3 protein including its structure and function was systematically examined by computer predictors to explicate its role in viral pathogenesis and drug resistance beyond its functions as accessory viral protein. Amino acid distribution showed ORF3 enrichment with disorder-promoting residues (Ala, Pro, Ser, Gly) while deficiency in order-promoting residues (Asn, Ile, Phe, Tyr and Trp). Initial investigation revealed ORF3 as IDP (entirely disordered protein) or IDPR (proteins consisting of IDRs with structured globular domains). Structural examination revealed preponderance of disordered regions interpreting ORF3 as moderately/highly disordered protein. Further disorder predictors categorized ORF3 as highly disordered protein/IDP. Identified sites and associated-crucial molecular functions revealed ORF3 involvement in diverse biological processes, substantiating them as targets of regulation. As ORF3 functions are yet to completely explored, thus, data on its disorderness could help in elucidating its disorder related functions.

Intrinsic Disorder in the Human Tear Proteome.

We aimed to characterize the proteome of human tears and assess for the presence of intrinsically disordered proteins (IDPs). IDPs, despite lacking a rigid three-dimensional structure, maintain biological functionality and could shed light on the molecular interactions within tears. We analyzed a dataset of 1475 proteins identified in the tear film of three healthy subjects. We employed several computational tools, including the Compositional Profiler, Rapid Intrinsic Disorder Analysis Online, Search Tool for the Retrieval of Interacting Genes, and Database of Disordered Protein Predictors to evaluate the intrinsic disorder, protein interactions, and functional characterization of the disordered regions within this proteome. Our analysis showed a notable inclination toward intrinsic disorder. Two out of 10 order-promoting residues and five out of 10 disorder-promoting residues were found enriched. Using the Predictor of Natural Disordered Regions (PONDR) VSL2 output, 95% of these proteins were classified as highly or moderately disordered. We revealed an extensive protein-protein interaction network with significant interaction enrichment. The most disordered proteins exhibited higher disorder binding sites and diverse posttranslational modifications compared to the most ordered ones. To the best of our knowledge, our study is the first comprehensive analysis of intrinsic disorder in the human tear film proteome, and it revealed an abundance of IDPs and their role in protein function and interaction networks. These findings suggest that variations in the intrinsic disorder of a tear film could be impacted by systemic and ocular conditions, offering promising avenues for disease biomarker identification and drug target development. Further research is needed to understand the implications of these findings in human health and disease.

A Trajectory of Discovery: Metabolic Regulation by the Conditionally Disordered Chloroplast Protein, CP12.

The chloroplast protein CP12, which is widespread in photosynthetic organisms, belongs to the intrinsically disordered proteins family. This small protein (80 amino acid residues long) presents a bias in its composition; it is enriched in charged amino acids, has a small number of hydrophobic residues, and has a high proportion of disorder-promoting residues. More precisely, CP12 is a conditionally disordered proteins (CDP) dependent upon the redox state of its four cysteine residues. During the day, reducing conditions prevail in the chloroplast, and CP12 is fully disordered. Under oxidizing conditions (night), its cysteine residues form two disulfide bridges that confer some stability to some structural elements. Like many CDPs, CP12 plays key roles, and its redox-dependent conditional disorder is important for the main function of CP12: the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO2 assimilation. Oxidized CP12 binds to glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase and thereby inhibits their activity. However, recent studies reveal that CP12 may have other functions beyond the CBB cycle regulation. In this review, we report the discovery of this protein, its features as a disordered protein, and the many functions this small protein can have.

The aqueous humor proteome is intrinsically disordered

•Our study demonstrated that intrinsic disorder is abundant in the aqueous humor.•The 749 aqueous proteins analyzed were enriched with disorder-promoting residues.•208 aqueous humor proteins were predicted to be highly intrinsically disordered.•Misregulation of IDPs may promote pathology in the aqueous humor.•IDPs in aqueous humor may serve as future targets for novel therapeutics.

Functional characterization of an unknown soybean intrinsically disordered protein in vitro and in Escherichia coli

Intrinsically disordered proteins (IDPs) possess a wide range of biological function in all organisms, however the specific functions of most IDPs are still unknown. Soybean LOC protein, LOC for short, is a heat-stable protein, which is more abundant in the stress-resistant radicles. Sequence alignment and phylogenetic analysis showed that LOC is a functionally unknown protein and conserved in Fabaceae. LOC, being enriched in most disorder-promoting residues and depleted in most order-promoting residues, was predicted to contain high levels of intrinsic disorder by several commonly used computational tools. However, it was also predicted to contain two disorder-based protein-protein binding sites and two short α-helical segments. The circular dichroism spectroscopic analysis showed that this protein is mostly disordered in water, but can form more α-helical structure in the presence of SDS and TFE. Functional in vitro studies showed that the LOC protein is able to prevent lactate dehydrogenase inactivation by freeze-thaw at a molar ratio of 10:1. Furthermore, in vivo analyses revealed the survival rate of Escherichia coli over-expressing LOC protein under the conditions of osmotic stress was noticeably increased in comparison with the control. These observations suggest that the intrinsically disordered protein LOC might serve as a chaperone and/or cell protector.

IDDomainSpotter: Compositional bias reveals domains in long disordered protein regions-Insights from transcription factors.

Protein domains constitute regions of distinct structural properties and molecular functions that are retained when removed from the rest of the protein. However, due to the lack of tertiary structure, the identification of domains has been largely neglected for long (>50 residues) intrinsically disordered regions. Here we present a sequence-based approach to assess and visualize domain organization in long intrinsically disordered regions based on compositional sequence biases. An online tool to find putative intrinsically disordered domains (IDDomainSpotter) in any protein sequence or sequence alignment using any particular sequence trait is available at http://www.bio.ku.dk/sbinlab/IDDomainSpotter. Using this tool, we have identified a putative domain enriched in hydrophilic and disorder-promoting residues (Pro, Ser, and Thr) and depleted in positive charges (Arg and Lys) bordering the folded DNA-binding domains of several transcription factors (p53, GCR, NAC46, MYB28, and MYB29). This domain, from two different MYB transcription factors, was characterized biophysically to determine its properties. Our analyses show the domain to be extended, dynamic and highly disordered. It connects the DNA-binding domain to other disordered domains and is present and conserved in several transcription factors from different families and domains of life. This example illustrates the potential of IDDomainSpotter to predict, from sequence alone, putative domains of functional interest in otherwise uncharacterized disordered proteins.

Sequence characteristics responsible for protein-protein interactions in the intrinsically disordered regions of caseins, amelogenins, and small heat-shock proteins.

Milk caseins and dental amelogenins are intrinsically disordered proteins (IDPs) that associate with themselves and others. Paradoxically, they are also described as hydrophobic proteins, which is difficult to reconcile with a solvent-exposed conformation. We attempt to resolve this paradox. We show that caseins and amelogenins are not hydrophobic proteins but they are more hydrophobic than most IDPs. Remarkably, uncharged residues from different regions of these mature proteins have a nearly constant average hydropathy but these regions exhibit different charged residue frequencies. A novel sequence analysis method was developed to identify hydrophobic and order-promoting regions that would favor conformational collapse. We found that such regions were uncommon; most hydrophobic and order-promoting residues were adjacent to hydrophilic or disorder-promoting residues. A further reason why caseins and amelogenins do not collapse is their high proportion of disorder-promoting proline residues. We conclude that in these proteins the hydrophobic effect is not large enough to cause conformational collapse but it can contribute, along with polar interactions, to protein-protein interactions. This behaviour is similar to the interaction of the disordered N-terminal region of small heat-shock proteins with either themselves during oligomer formation or other, unfolding, proteins during chaperone action.

On the relationship between the conserved ‘black’ and ‘gray’ structural clusters and intrinsic disorder in parvalbumins

Recently we found two highly conserved structural motifs in the members of the EF-hand protein family, which provide a supporting scaffold for their Ca2+ binding loops. Each structural motif is formed by a cluster of three amino acids. These clusters were called ‘black’ cluster (cluster I) and ‘gray’ cluster (cluster II). In the present work, we studied the relationship between the location of the ‘black’ and ‘gray’ structural clusters in parvalbumins and the location of the amino acid sequence regions with a tendency for intrinsic disorder. This analysis revealed that in parvalbumins, the residues in the vicinity of the conserved structural clusters constitute parts of the conserved motifs enriched in the disorder-promoting residues. Therefore, the clusters found in parvalbumins are characterized not only by the presence of conserved amino acid residues, but also by the conserved distribution of the intrinsic disorder predisposition within their sequences, suggesting the presence of conserved structural dynamics in the apo-forms of parvalbumins, where the black cluster appears to have greater mobility than the gray cluster.

The IDP-Specific Force Field ff14IDPSFF Improves the Conformer Sampling of Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) or intrinsically disordered regions do not have a fixed tertiary structure but play key roles in signal regulation, molecule recognition, and drug targeting. However, it is difficult to study the structure and function of IDPs by traditional experimental methods because of their diverse conformations. Limitations of current generic protein force fields and solvent models were reported in the previous simulations of IDPs. We have also explored overcoming these limitations by developing the ff99IDPs and ff14IDPs force fields to correct the dihedral distribution for eight disorder-promoting residues often observed in IDPs and found encouraging improvements. Here we extend our correction of backbone dihedral terms to all 20 naturally occurring amino acids in the IDP-specific force field ff14IDPSFF to further improve the quality of the modeling of IDPs. Extensive tests of seven IDPs and 14 unstructured short peptides show that the simulated Cα chemical shifts obtained with the ff14IDPSFF force field are in quantitative agreement with those from NMR experiments and are more accurate than those obtained with the base generic force field and also our previous ff14IDPs that only corrects the eight disorder-promoting amino acids. The influence of the solvent model was also investigated and found to be less important. Finally, our explicit-solvent MD simulations further show that ff14IDPSFF can still be used to model structural and dynamical properties of two tested folded proteins, with a slightly better agreement in the loop regions for both structural and dynamical properties. These findings confirm that the newly developed IDP-specific force field ff14IDPSFF can improve the conformer sampling of intrinsically disordered proteins.

Molecular Dynamics Simulations of Intrinsically Disordered Proteins: On the Accuracy of the TIP4P-D Water Model and the Representativeness of Protein Disorder Models.

Here, we first present a follow-up to a previous work by our group on the problematic of molecular dynamics simulations of intrinsically disordered proteins (IDPs) [ Henriques et al. J. Chem. Theory Comput. 2015 , 11 , 3420 - 3431 ], using the recently developed TIP4P-D water model. When used in conjunction with the standard AMBER ff99SB-ILDN force field and applied to the simulation of Histatin 5, our IDP model, we obtain results which are in excellent agreement with the best performing IDP-suitable force field from the earlier study and with experiment. We then assess the representativeness of the IDP models used in these and similar studies, finding that most are too short in comparison to the average IDP and contain a bias toward hydrophilic amino acid residues. Moreover, several key order- and disorder-promoting residues are also found to be misrepresented. It seems appropriate for future studies to address these issues.

Casein and casein micelle structures, functions and diversity in 20 species

Primary structures of caseins from 20 species, including two monotremes and two marsupials, have been compared. Sequences of the mature proteins are very divergent, whereas variation in amino acid composition is mostly restricted to a range of disorder-promoting residues. The number and size of clusters of phosphorylation sites in the caseins is variable, blurring the boundaries between them. Casein polar tract sequences were found in all caseins, though of variable lengths, and are chiefly responsible for weak and dynamic interactions among the tangled web of peptide chains in the matrix of casein micelles. The interactions take the predominant form of backbone-to-backbone contacts rather than the sequence-specific side chain interactions of the hydrophobic effect. It is suggested that the dynamic casein micelle matrix be represented by an ensemble of interchanging structures with different types and degrees of inhomogeneity, influenced by solvent quality and other environmental factors.

Duplication of the V3 domain in hepatitis C virus (1b) NS5A protein: Clonal analysis and physicochemical properties related to hepatocellular carcinoma occurrence

BackgroundHepatitis C virus non-structural protein 5A is known to play a role in development of hepatocellular carcinoma (HCC) via interactions with host cell pathways. ObjectivesHepatitis C virus genotype 1b strains presenting a wide insertion of 31 amino acids in the non-structural protein 5A V3 domain (V3 DI) were studied to determine whether this V3-like additional domain (V3 DII) was associated with HCC occurrence. Study designSeventy-four patients’ sera were screened for V3 DII presence regarding clinical status. ResultsThree strains with duplicated V3 were detected among patients with progression to HCC (n=28), two strains among patients with liver cirrhosis (Ci, n=27) and none among patients with chronic hepatitis (Chr, n=19). Phylogenetic trees built from V3 DI and V3 DII sequences indicated that the latter clustered separately. In between-group clonal analysis, V3 DII sequences from the HCC group were found to be more distant from HCV-J than V3 DI sequences (p<0.0001). Between-group comparisons showed significant differences in genetic distances from HCV-J, in HCC V3 DI and HCC V3 DII compared to Ci V3 DI and Ci V3 DII sequences (p<0.0001). HCC V3 DII domain and its junction with V3 DI exhibited higher Shannon entropy values and enrichment in disorder-promoting residues. ConclusionsTaken together, our results suggest that V3 DII evolution may differ in strains associated with HCC occurrence. The presence of an intrinsically “disordered” V3 duplicate may alter the NS5A protein network. Further investigations are necessary to elucidate the potential impact of V3 duplication in the context of carcinogenesis.

Test and Evaluation of ff99IDPs Force Field for Intrinsically Disordered Proteins.

Over 40% of eukaryotic proteomic sequences have been predicted to be intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) and confirmed to be associated with many diseases. However, widely used force fields cannot well reproduce the conformers of IDPs. Previously the ff99IDPs force field was released to simulate IDPs with CMAP energy corrections for the eight disorder-promoting residues. In order to further confirm the performance of ff99IDPs, three representative IDP systems (arginine-rich HIV-1 Rev, aspartic proteinase inhibitor IA3, and α-synuclein) were used to test and evaluate the simulation results. The results show that for free disordered proteins, the chemical shifts from the ff99IDPs simulations are in quantitative agreement with those from reported NMR measurements and better than those from ff99SBildn. Thus, ff99IDPs can sample more clusters of disordered conformers than ff99SBildn. For structural proteins, both ff99IDPs and ff99SBildn can well reproduce the conformations. In general, ff99IDPs can successfully be used to simulate the conformations of IDPs and IDRs in both bound and free states. However, relative errors could still be found at the boundaries of ordered residues scattered in long disorder-promoting sequences. Therefore, polarizable force fields might be one of the possible ways to further improve the performance on IDPs.

The intrinsic disorder alphabet. III. Dual personality of serine.

Proteins are natural polypeptides consisting of 20 major amino acid residues, content and order of which in a given amino acid sequence defines the ability of a related protein to fold into unique functional state or to stay intrinsically disordered. Amino acid sequences code for both foldable (ordered) proteins/domains and for intrinsically disordered proteins (IDPs) and IDP regions (IDPRs), but these sequence codes are dramatically different. This difference starts with a very general property of the corresponding amino acid sequences, namely, their compositions. IDPs/IDPRs are enriched in specific disorder-promoting residues, whereas amino acid sequences of ordered proteins/domains typically contain more order-promoting residues. Therefore, the relative abundances of various amino acids in ordered and disordered proteins can be used to scale amino acids according to their disorder promoting potentials. This review continues a series of publications on the roles of different amino acids in defining the phenomenon of protein intrinsic disorder and represents serine, which is the third most disorder-promoting residue. Similar to previous publications, this review represents some physico-chemical properties of serine and the roles of this residue in structures and functions of ordered proteins, describes major posttranslational modifications tailored to serine, and finally gives an overview of roles of serine in structure and functions of intrinsically disordered proteins.

New Force Field on Modeling Intrinsically Disordered Proteins

Intrinsically disordered proteins or intrinsically disordered protein regions comprise a large portion of eukaryotic proteomes (between 35% and 51%). These intrinsically disordered proteins were found to link with cancer and various other diseases. However, widely used additive force field parameter sets are insufficient in quantifying the structural properties of intrinsically disordered proteins. Therefore, we explored to a systematic correction of a base additive force field parameter set (chosen as Amber ff99SBildn) to correct the biases that was first demonstrated in simulations with the base parameter set. Specifically, the φ/ψ distributions of disorder-promoting residues were systematically corrected with the CMAP method. Our simulations show that the CMAP corrected Amber parameter set, termed ff99IDPs, improves the φ/ψ distributions of the disorder-promoting residues with respect to the benchmark data of intrinsically disordered protein structures, with root mean-squared percentage deviation less than 0.15% between the simulation and the benchmark. Our further validation shows that the chemical shifts from the ff99IDPs simulations are in quantitative agreement with those from reported NMR measurements for two tested IDPs, MeV NTAIL , and p53. The predicted residue dipolar couplings also show high correlation with experimental data. Interestingly, our simulations show that ff99IDPs can still be used to model the ordered state when the intrinsically disordered proteins are in complex, in contrast to ff99SBildn that can be applied well only to the ordered complex structures. These findings confirm that the newly proposed Amber ff99IDPs parameter set provides a reasonable tool in further studies of intrinsically disordered protein structures. In addition, our study also shows the importance of considering intrinsically disordered protein structures in general-purposed force field developments for both additive and non-additive models.

The alphabet of intrinsic disorder: II. Various roles of glutamic acid in ordered and intrinsically disordered proteins.

The ability of a protein to fold into unique functional state or to stay intrinsically disordered is encoded in its amino acid sequence. Both ordered and intrinsically disordered proteins (IDPs) are natural polypeptides that use the same arsenal of 20 proteinogenic amino acid residues as their major building blocks. The exceptional structural plasticity of IDPs, their capability to exist as heterogeneous structural ensembles and their wide array of important disorder-based biological functions that complements functional repertoire of ordered proteins are all rooted within the peculiar differential usage of these building blocks by ordered proteins and IDPs. In fact, some residues (so-called disorder-promoting residues) are noticeably more common in IDPs than in sequences of ordered proteins, which, in their turn, are enriched in several order-promoting residues. Furthermore, residues can be arranged according to their “disorder promoting potencies,” which are evaluated based on the relative abundances of various amino acids in ordered and disordered proteins. This review continues a series of publications on the roles of different amino acids in defining the phenomenon of protein intrinsic disorder and concerns glutamic acid, which is the second most disorder-promoting residue.

The alphabet of intrinsic disorder: I. Act like a Pro: On the abundance and roles of proline residues in intrinsically disordered proteins.

A significant fraction of every proteome is occupied by biologically active proteins that do not form unique three-dimensional structures. These intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) have essential biological functions and are characterized by extensive structural plasticity. Such structural and functional behavior is encoded in the amino acid sequences of IDPs/IDPRs, which are enriched in disorder-promoting residues and depleted in order-promoting residues. In fact, amino acid residues can be arranged according to their disorder-promoting tendency to form an alphabet of intrinsic disorder that defines the structural complexity and diversity of IDPs/IDPRs. This review is the first in a series of publications dedicated to the roles that different amino acid residues play in defining the phenomenon of protein intrinsic disorder. We start with proline because data suggests that of the 20 common amino acid residues, this one is the most disorder-promoting.

An intrinsically disordered protein, CP12: jack of all trades and master of the Calvin cycle

Many proteins contain disordered regions under physiological conditions and lack specific three-dimensional structure. These are referred to as IDPs (intrinsically disordered proteins). CP12 is a chloroplast protein of approximately 80 amino acids and has a molecular mass of approximately 8.2-8.5kDa. It is enriched in charged amino acids and has a small number of hydrophobic residues. It has a high proportion of disorder-promoting residues, but has at least two (often four) cysteine residues forming one (or two) disulfide bridge(s) under oxidizing conditions that confers some order. However, CP12 behaves like an IDP. It appears to be universally distributed in oxygenic photosynthetic organisms and has recently been detected in a cyanophage. The best studied role of CP12 is its regulation of the Calvin cycle responsible for CO2 assimilation. Oxidized CP12 forms a supramolecular complex with two key Calvin cycle enzymes, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and PRK (phosphoribulokinase), down-regulating their activity. Association-dissociation of this complex, induced by the redox state of CP12, allows the Calvin cycle to be inactive in the dark and active in the light. CP12 is promiscuous and interacts with other enzymes such as aldolase and malate dehydrogenase. It also plays other roles in plant metabolism such as protecting GAPDH from inactivation and scavenging metal ions such as copper and nickel, and it is also linked to stress responses. Thus CP12 seems to be involved in many functions in photosynthetic cells and behaves like a jack of all trades as well as being a master of the Calvin cycle.

The role of protein disorder in the 14-3-3 interaction network

Disordered regions are segments of a protein that do not fold completely and thus remain flexible. These regions have key physiological roles, particularly in phospho-proteins, which are enriched in disorder-promoting residues surrounding their phosphorylation sites. 14-3-3 proteins are ordered hubs that interact with multiple and diverse intrinsically disordered phosphorylated targets. This provides 14-3-3 with the ability to participate in and to regulate multiple signalling networks. Here, I review the effect of structural disorder on the mechanism involved in 14-3-3 protein-protein interactions and how 14-3-3 impacts cell biology through disordered ligands. How 14-3-3 proteins constitute an advantageous system to identify novel classes of biological tools is discussed with a special emphasis on a particular-and innovative-use of small molecules to stabilize 14-3-3 protein complexes, useful to study gene expression, cancer signalling and neurodegenerative diseases.

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.