Evolutionary rate heterogeneity in proteins with long disordered regions

The dominant view in protein science is that a three-dimensional (3-D) structure is a prerequisite for protein function. In contrast to this dominant view, there are many counterexample proteins that fail to fold into a 3-D structure, or that have local regions that fail to fold, and yet carry out function. Protein without fixed 3-D structure is called intrinsically disordered. Motivated by anecdotal accounts of higher rates of sequence evolution in disordered protein than in ordered protein we are exploring the molecular evolution of disordered proteins. To test whether disordered protein evolves more rapidly than ordered protein, pairwise genetic distances were compared between the ordered and the disordered regions of 26 protein families having at least one member with a structurally characterized region of disorder of 30 or more consecutive residues. For five families, there were no significant differences in pairwise genetic distances between ordered and disordered sequences. The disordered region evolved significantly more rapidly than the ordered region for 19 of the 26 families. The functions of these disordered regions are diverse, including binding sites for protein, DNA, or RNA and also including flexible linkers. The functions of some of these regions are unknown. The disordered regions evolved significantly more slowly than the ordered regions for the two remaining families. The functions of these more slowly evolving disordered regions include sites for DNA binding. More work is needed to understand the underlying causes of the variability in the evolutionary rates of intrinsically ordered and disordered protein.     

Markov models of amino acid substitution to study proteins with intrinsically disordered regions

Background

Intrinsically disordered proteins (IDPs) or proteins with disordered regions (IDRs) do not have a well-defined tertiary structure, but perform a multitude of functions, often relying on their native disorder to achieve the binding flexibility through changing to alternative conformations. Intrinsic disorder is frequently found in all three kingdoms of life, and may occur in short stretches or span whole proteins. To date most studies contrasting the differences between ordered and disordered proteins focused on simple summary statistics. Here, we propose an evolutionary approach to study IDPs, and contrast patterns specific to ordered protein regions and the corresponding IDRs.

Results

Two empirical Markov models of amino acid substitutions were estimated, based on a large set of multiple sequence alignments with experimentally verified annotations of disordered regions from the DisProt database of IDPs. We applied new methods to detect differences in Markovian evolution and evolutionary rates between IDRs and the corresponding ordered protein regions. Further, we investigated the distribution of IDPs among functional categories, biochemical pathways and their preponderance to contain tandem repeats.

Conclusions

We find significant differences in the evolution between ordered and disordered regions of proteins. Most importantly we find that disorder promoting amino acids are more conserved in IDRs, indicating that in some cases not only amino acid composition but the specific sequence is important for function. This conjecture is also reinforced by the observation that for of our data set IDRs evolve more slowly than the ordered parts of the proteins, while we still support the common view that IDRs in general evolve more quickly. The improvement in model fit indicates a possible improvement for various types of analyses e.g. de novo disorder prediction using a phylogenetic Hidden Markov Model based on our matrices showed a performance similar to other disorder predictors.     

Ensembles from Ordered and Disordered Proteins Reveal Similar Structural Constraints during Evolution

The conformations accessible to proteins are determined by the inter-residue interactions between amino acid residues. During evolution, structural constraints that are required for protein function providing biologically relevant information can exist. Here, we studied the proportion of sites evolving under structural constraints in two very different types of ensembles, those coming from ordered and disordered proteins. Using a structurally constrained model of protein evolution, we found that both types of ensembles show comparable, near 40%, number of positions evolving under structural constraints. Among these sites, ~ 68% are in disordered regions and ~ 57% of them show long-range inter-residue contacts. Also, we found that disordered ensembles are redundant in reference to their structurally constrained evolutionary information and could be described on average with ~ 11 conformers. Despite the different complexity of the studied ensembles and proteins, the similar constraints reveal a comparable level of selective pressure to maintain their biological functions. These results highlight the importance of the evolutionary information to recover meaningful biological information to further characterize conformational ensembles.     

Long indels are disordered: A study of disorder and indels in homologous eukaryotic proteins

Proteins evolve through point mutations as well as by insertions and deletions (indels). During the last decade it has become apparent that protein regions that do not fold into three-dimensional structures, i.e. intrinsically disordered regions, are quite common. Here, we have studied the relationship between protein disorder and indels using HMM–HMM pairwise alignments in two sets of orthologous eukaryotic protein pairs. First, we show that disordered residues are much more frequent among indel residues than among aligned residues and, also are more prevalent among indels than in coils. Second, we observed that disordered residues are particularly common in longer indels. Disordered indels of short-to-medium size are prevalent in the non-terminal regions of proteins while the longest indels, ordered and disordered alike, occur toward the termini of the proteins where new structural units are comparatively well tolerated. Finally, while disordered regions often evolve faster than ordered regions and disorder is common in indels, there are some previously recognized protein families where the disordered region is more conserved than the ordered region. We find that these rare proteins are often involved in information processes, such as RNA processing and translation. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

Serine/arginine-rich splicing factors belong to a class of intrinsically disordered proteins

Serine/arginine-rich (SR) splicing factors play an important role in constitutive and alternative splicing as well as during several steps of RNA metabolism. Despite the wealth of functional information about SR proteins accumulated to-date, structural knowledge about the members of this family is very limited. To gain a better insight into structure-function relationships of SR proteins, we performed extensive sequence analysis of SR protein family members and combined it with ordered/disordered structure predictions. We found that SR proteins have properties characteristic of intrinsically disordered (ID) proteins. The amino acid composition and sequence complexity of SR proteins were very similar to those of the disordered protein regions. More detailed analysis showed that the SR proteins, and their RS domains in particular, are enriched in the disorder-promoting residues and are depleted in the order-promoting residues as compared to the entire human proteome. Moreover, disorder predictions indicated that RS domains of SR proteins were completely unstructured. Two different classification methods, the charge-hydropathy measure and the cumulative distribution function (CDF) of the disorder scores, were in agreement with each other, and they both strongly predicted members of the SR protein family to be disordered. This study emphasizes the importance of the disordered structure for several functions of SR proteins, such as for spliceosome assembly and for interaction with multiple partners. In addition, it demonstrates the usefulness of order/disorder predictions for inferring protein structure from sequence.     

Using Bayesian multinomial classifier to predict whether a given protein sequence is intrinsically disordered

Intrinsically disordered proteins (IDPs) lack a well-defined three-dimensional structure under physiological conditions. Intrinsic disorder is a common phenomenon, particularly in multicellular eukaryotes, and is responsible for important protein functions including regulation and signaling. Many disease-related proteins are likely to be intrinsically disordered or to have disordered regions. In this paper, a new predictor model based on the Bayesian classification methodology is introduced to predict for a given protein or protein region if it is intrinsically disordered or ordered using only its primary sequence. The method allows to incorporate length-dependent amino acid compositional differences of disordered regions by including separate statistical representations for short, middle and long disordered regions. The predictor was trained on the constructed data set of protein regions with known structural properties. In a Jack-knife test, the predictor achieved the sensitivity of 89.2% for disordered and 81.4% for ordered regions. Our method outperformed several reported predictors when evaluated on the previously published data set of Prilusky et al. [2005. FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 21 (16), 3435-3438]. Further strength of our approach is the ease of implementation.     

Do sequence neighbours of intrinsically disordered regions promote structural flexibility in intrinsically disordered proteins?

Intrinsically disordered proteins (IDPs) are crucial players in various cellular activities. Several experimental and computational analyses have been conducted to study structural pliability and functional potential of IDPs. In spite of active research in past few decades, what induces structural disorder in IDPs and how is still elusive. Many studies testify that sequential and spatial neighbours often play important roles in determining structural and functional behaviour of proteins. Considering this fact, we assessed sequence neighbours of intrinsically disordered regions (IDRs) to understand if they have any role to play in inducing structural flexibility in IDPs. Our analysis includes 97% eukaryotic IDPs and 3% from bacteria and viruses. Physicochemical and structural parameters including amino acid propensity, hydrophobicity, secondary structure propensity, relative solvent accessibility, B-factor and atomic packing density are used to characterise the neighbouring residues of IDRs (NRIs). We show that NRIs exhibit a unique nature, which makes them stand out from both ordered and disordered residues. They show correlative occurrences of residue pairs like Ser-Thr and Gln-Asn, indicating their tendency to avoid strong biases of order or disorder promoting amino acids. We also find differential preferences of amino acids between N- and C-terminal neighbours, which might indicate a plausible directional effect on the dynamics of adjacent IDRs. We designed an efficient prediction tool using Random Forest to distinguish the NRIs from the ordered residues. Our findings will contribute to understand the behaviour of IDPs, and may provide potential lead in deciphering the role of IDRs in protein folding and assembly.     

Unusual biophysics of intrinsically disordered proteins

Research of a past decade and a half leaves no doubt that complete understanding of protein functionality requires close consideration of the fact that many functional proteins do not have well-folded structures. These intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered protein regions (IDPRs) are highly abundant in nature and play a number of crucial roles in a living cell. Their functions, which are typically associated with a wide range of intermolecular interactions where IDPs possess remarkable binding promiscuity, complement functional repertoire of ordered proteins. All this requires a close attention to the peculiarities of biophysics of these proteins. In this review, some key biophysical features of IDPs are covered. In addition to the peculiar sequence characteristics of IDPs these biophysical features include sequential, structural, and spatiotemporal heterogeneity of IDPs; their rough and relatively flat energy landscapes; their ability to undergo both induced folding and induced unfolding; the ability to interact specifically with structurally unrelated partners; the ability to gain different structures at binding to different partners; and the ability to keep essential amount of disorder even in the bound form. IDPs are also characterized by the “turned-out” response to the changes in their environment, where they gain some structure under conditions resulting in denaturation or even unfolding of ordered proteins. It is proposed that the heterogeneous spatiotemporal structure of IDPs/IDPRs can be described as a set of foldons, inducible foldons, semi-foldons, non-foldons, and unfoldons. They may lose their function when folded, and activation of some IDPs is associated with the awaking of the dormant disorder. It is possible that IDPs represent the “edge of chaos” systems which operate in a region between order and complete randomness or chaos, where the complexity is maximal. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

Influence of Sequence Changes and Environment on Intrinsically Disordered Proteins

Many large-scale studies on intrinsically disordered proteins are implicitly based on the structural models deposited in the Protein Data Bank. Yet, the static nature of deposited models supplies little insight into variation of protein structure and function under diverse cellular and environmental conditions. While the computational predictability of disordered regions provides practical evidence that disorder is an intrinsic property of proteins, the robustness of disordered regions to changes in sequence or environmental conditions has not been systematically studied. We analyzed intrinsically disordered regions in the same or similar proteins crystallized independently and studied their sensitivity to changes in protein sequence and parameters of crystallographic experiments. The observed changes in the existence, position, and length of disordered regions indicate that their appearance in X-ray structures dramatically depends on changes in amino acid sequence and peculiarities of the crystallographic experiment. Our study also raises general questions regarding protein evolution and the regulation of protein structure, dynamics, and function via variations in cellular and environmental conditions.     

A practical overview of protein disorder prediction methods

In the past few years there has been a growing awareness that a large number of proteins contain long disordered (unstructured) regions that often play a functional role. However, these disordered regions are still poorly detected. Recognition of disordered regions in a protein is important for two main reasons: reducing bias in sequence similarity analysis by avoiding alignment of disordered regions against ordered ones, and helping to delineate boundaries of protein domains to guide structural and functional studies. As none of the available method for disorder prediction can be taken as fully reliable on its own, we present an overview of the methods currently employed highlighting their advantages and drawbacks. We show a few practical examples of how they can be combined to avoid pitfalls and to achieve more reliable predictions.     

Disorder transitions and conformational diversity cooperatively modulate biological function in proteins

Structural differences between conformers sustain protein biological function. Here, we studied in a large dataset of 745 intrinsically disordered proteins, how ordered‐disordered transitions modulate structural differences between conformers as derived from crystallographic data. We found that almost 50% of the proteins studied show no transitions and have low conformational diversity while the rest show transitions and a higher conformational diversity. In this last subset, 60% of the proteins become more ordered after ligand binding, while 40% more disordered. As protein conformational diversity is inherently connected with protein function our analysis suggests differences in structure‐function relationships related to order‐disorder transitions.     

Intrinsically disordered regions of p53 family are highly diversified in evolution

Proteins of the p53 family are expressed in vertebrates and in some invertebrate species. The main function of these proteins is to control and regulate cell cycle in response to various cellular signals, and therefore to control the organism's development. The regulatory functions of the p53 family members originate mostly from their highly-conserved and well-structured DNA-binding domains. Many human diseases (including various types of cancer) are related to the missense mutations within this domain. The ordered DNA-binding domains of the p53 family members are surrounded by functionally important intrinsically disordered regions. In this study, substitution rates and propensities in different regions of p53 were analyzed. The analyses revealed that the ordered DNA-binding domain is conserved, whereas disordered regions are characterized by high sequence diversity. This diversity was reflected both in the number of substitutions and in the types of substitutions to which each amino acid was prone. These results support the existence of a positive correlation between protein intrinsic disorder and sequence divergence during the evolutionary process. This higher sequence divergence provides strong support for the existence of disordered regions in p53 in vivo for if they were structured, they would evolve at similar rates as the rest of the protein.     

Comparative Laboratory Evolution of Ordered and Disordered Enzymes

Intrinsically disordered proteins are ubiquitous in nature. To assess potential evolutionary advantages and disadvantages of structural disorder under controlled laboratory conditions, we directly compared the evolvability of weakly active ordered and disordered variants of dihydrofolate reductase by genetic selection. The circularly permuted Escherichia coli enzyme, which exists as a molten globule in the absence of ligands, and a well folded deletion mutant of the Bacillus stearothermophilus enzyme served as starting points. Both scaffolds evolved at similar rates and to similar extents, reaching near-native activity after three rounds of mutagenesis and selection. Surprisingly, however, the starting structural properties of the two scaffolds changed only marginally during optimization. Although the ordered and disordered proteins accumulated distinct sets of mutations, the changes introduced likely improved catalytic efficiency indirectly in both cases by bolstering the network of dynamic conformational fluctuations that productively couple into the reaction coordinate.     

Intrinsic disorder in the human spliceosomal proteome

The spliceosome is a molecular machine that performs the excision of introns from eukaryotic pre-mRNAs. This macromolecular complex comprises in human cells five RNAs and over one hundred proteins. In recent years, many spliceosomal proteins have been found to exhibit intrinsic disorder, that is to lack stable native three-dimensional structure in solution. Building on the previous body of proteomic, structural and functional data, we have carried out a systematic bioinformatics analysis of intrinsic disorder in the proteome of the human spliceosome. We discovered that almost a half of the combined sequence of proteins abundant in the spliceosome is predicted to be intrinsically disordered, at least when the individual proteins are considered in isolation. The distribution of intrinsic order and disorder throughout the spliceosome is uneven, and is related to the various functions performed by the intrinsic disorder of the spliceosomal proteins in the complex. In particular, proteins involved in the secondary functions of the spliceosome, such as mRNA recognition, intron/exon definition and spliceosomal assembly and dynamics, are more disordered than proteins directly involved in assisting splicing catalysis. Conserved disordered regions in spliceosomal proteins are evolutionarily younger and less widespread than ordered domains of essential spliceosomal proteins at the core of the spliceosome, suggesting that disordered regions were added to a preexistent ordered functional core. Finally, the spliceosomal proteome contains a much higher amount of intrinsic disorder predicted to lack secondary structure than the proteome of the ribosome, another large RNP machine. This result agrees with the currently recognized different functions of proteins in these two complexes.     

Introducing intrinsic disorder reduces electrostatic steering in protein-protein interactions

Protein-protein interactions underlie many critical biology functions, such as cellular signaling and gene expression, in which electrostatic interactions can play a critical role in mediating the specificity and stability of protein complexes. A substantial portion of proteins are intrinsically disordered, and the influences of structural disorder on binding kinetics and thermodynamics have been widely investigated. However, whether the effect of electrostatic steering depends on structural disorder remains unexplored. In this work, we addressed the consequence of introducing intrinsic disorder in the electrostatic steering of the E3/Im3 complex using molecular dynamics simulation. Our results recapitulated the experimental observations that the responses of stability and kinetics to salt concentration for the ordered E3/Im3 complex were larger than those for the disordered E3/Im3 complex. Mechanistic analysis revealed that the native contact interactions involved in the encounter state and the transition state were essentially identical for both ordered and disordered E3. Therefore, the observed difference in electrostatic steering between ordered E3 and disordered E3 may result from their difference in conformation rather than their difference in binding mechanism. Because charged residues are frequently involved in protein-protein interactions, our results suggest that increasing structural disorder is expected to generally modulate the effect of electrostatic steering.     

Predicting mostly disordered proteins by using structure-unknown protein data

Background  

Predicting intrinsically disordered proteins is important in structural biology because they are thought to carry out various cellular functions even though they have no stable three-dimensional structure. We know the structures of far more ordered proteins than disordered proteins. The structural distribution of proteins in nature can therefore be inferred to differ from that of proteins whose structures have been determined experimentally. We know many more protein sequences than we do protein structures, and many of the known sequences can be expected to be those of disordered proteins. Thus it would be efficient to use the information of structure-unknown proteins in order to avoid training data sparseness. We propose a novel method for predicting which proteins are mostly disordered by using spectral graph transducer and training with a huge amount of structure-unknown sequences as well as structure-known sequences.     

Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry

Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three‐dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of ¹H/²H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N‐ and C‐terminal tails were evaluated using ¹H‐¹⁵N HSQC and ¹H‐¹⁵N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS‐based truncated construct for a 77‐residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Between order and disorder in protein structures: analysis of "dual personality" fragments in proteins

In their natural environment, three-dimensional structures of proteins undergo significant fluctuations and are often partially or completely disordered. This phenomenon recently became the focus of much attention, as many proteins, especially from higher organisms, were shown to contain large intrinsically disordered regions. Such disordered regions may become ordered only under very specific circumstances, if at all, and can be recognized by specific amino acid composition and sequence signatures. Here, we suggest that the balance between order and disorder is much more subtle in that many regions are very close to the order/disorder boundary. Specifically, analysis of redundant sets of experimental models of protein structures, where emphasis is put on comparison of structures of identical proteins solved in different conditions and functional states, shows hundreds of fragments captured in two states: ordered and disordered. We show that such fragments, which we call here "dual personality" (DP) fragments, have distinctive features that differentiate them from both regularly folded and intrinsically disordered fragments. We hypothesize, and show on several examples, that such fragments are often targets of regulation, either by allostery or posttranslational modifications.