Analysis of ordered and disordered protein complexes reveals structural features discriminating between stable and unstable monomers

Most proteins exist in the cell as multi-component assemblies. However, which proteins need to be present simultaneously in order to perform a given function is frequently unknown. The first step toward this goal would be to predict proteins that can function only when in a complexed form. Here, we propose a scheme to distinguish whether the protein components are ordered (stable) or disordered when separated from their complexed partners. We analyze structural characteristics of several types of complexes, such as natively unstructured proteins, ribosomal proteins, two-state and three-state complexes, and crystal-packing dimers. Our analysis makes use of the fact that natively unstructured proteins, which undergo a disorder-to-order transition upon binding their partner, and stable monomeric proteins, which exist as dimers only in their crystal form, provide examples of two vastly different scenarios. We find that ordered monomers can be distinguished from disordered monomers on the basis of the per-residue surface and interface areas, which are significantly smaller for ordered proteins. With this scale, two-state dimers (where the monomers unfold upon dimer separation) and ribosomal proteins are shown to resemble disordered proteins. On the other hand, crystal-packing dimers, whose monomers are stable in solution, fall into the ordered protein category. While there should be a continuum in the distributions, nevertheless, the per-residue scale measures the confidence in the determination of whether a protein can exist as a stable monomer. Further analysis, focusing on the chemical and contact preferences at the interface, interior and exposed surface areas, reveals that disordered proteins lack a strong hydrophobic core and are composed of highly polar surface area. We discuss the implication of our results for de novo design of stable monomeric proteins and peptides.     

Intrinsically disordered regions in autophagy proteins

Autophagy is an essential eukaryotic pathway required for cellular homeostasis. Numerous key autophagy effectors and regulators have been identified, but the mechanism by which they carry out their function in autophagy is not fully understood. Our rigorous bioinformatic analysis shows that the majority of key human autophagy proteins include intrinsically disordered regions (IDRs), which are sequences lacking stable secondary and tertiary structure; suggesting that IDRs play an important, yet hitherto uninvestigated, role in autophagy. Available crystal structures corroborate the absence of structure in some of these predicted IDRs. Regions of orthologs equivalent to the IDRs predicted in the human autophagy proteins are poorly conserved, indicating that these regions may have diverse functions in different homologs. We also show that IDRs predicted in human proteins contain several regions predicted to facilitate protein–protein interactions, and delineate the network of proteins that interact with each predicted IDR‐containing autophagy protein, suggesting that many of these interactions may involve IDRs. Lastly, we experimentally show that a BCL2 homology 3 domain (BH3D), within the key autophagy effector BECN1 is an IDR. This BH3D undergoes a dramatic conformational change from coil to α‐helix upon binding to BCL2s, with the C‐terminal half of this BH3D constituting a binding motif, which serves to anchor the interaction of the BH3D to BCL2s. The information presented here will help inform future in‐depth investigations of the biological role and mechanism of IDRs in autophagy proteins. Proteins 2014; 82:565–578. © 2013 Wiley Periodicals, Inc.     

Computational analysis of position-dependent disorder content in DisProt database

A bioinformatics analysis of disorder content of proteins from the DisProt database has been performed with respect to position of disordered residues.Each protein chain was divided into three parts:N-and C-terminal parts with each containing 30 amino acid(AA) residues and the middle region containing the remaining AA residues.The results show that in terminal parts,the percentage of disordered AA residues is higher than that of all AA residues(17% of disordered AA residues and 11% of all).We analyzed the percentage of disorder for each of 20 AA residues in the three parts of proteins with respect to their hydropathy and molecular weight.For each AA,the percentage of disorder in the middle part is lower than that in terminal parts which is comparable at the two termini.A new scale of AAs has been introduced according to their disorder content in the middle part of proteins:CIFWMLYHRNVTAGQDSKEP.All big hydrophobic AAs are less frequently disordered,while almost all small hydrophilic AAs are more frequently disordered.The results obtained may be useful for construction and improving predictors for protein disorder.     

Functional dissection of an intrinsically disordered protein: Understanding the roles of different domains of Knr4 protein in protein�Cprotein interactions

Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, participates in cell wall formation and cell cycle regulation. It is constituted of a functional central globular core flanked by a poorly structured N‐terminal and large natively unfolded C‐terminal domains. Up to now, about 30 different proteins have been reported to physically interact with Knr4. Here, we used an in vivo two‐hybrid system approach and an in vitro surface plasmon resonance (BIAcore) technique to compare the interaction level of different Knr4 deletion variants with given protein partners. We demonstrate the indispensability of the N‐terminal domain of Knr4 for the interactions. On the other hand, presence of the unstructured C‐terminal domain has a negative effect on the interaction strength. In protein interactions networks, the most highly connected proteins or “hubs” are significantly enriched in unstructured regions, and among them the transient hub proteins contain the largest and most highly flexible regions. The results presented here of our analysis of Knr4 protein suggest that these large disordered regions are not always involved in promoting the protein–protein interactions of hub proteins, but in some cases, might rather inhibit them. We propose that this type of regions could prevent unspecific protein interactions, or ensure the correct timing of occurrence of transient interactions, which may be of crucial importance for different signaling and regulation processes.     

Structural disorder: a tool for housekeeping proteins performing tissue-specific interactions

An interaction between a pair of proteins unique for a particular tissue is denoted as a tissue-specific interaction (TSI). Tissue-specific (TS) proteins always perform TSIs with a limited number of interacting partners. However, it has been claimed that housekeeping (HK) proteins frequently take part in TSIs. This is actually an unusual phenomenon. How a single HK protein mediates TSIs – remains an interesting yet an unsolved question. We have hypothesized that HK proteins have attained a high degree of structural flexibility to modulate TSIs efficiently. We have observed that HK proteins are selected to be intrinsically disordered compared to TS proteins. Therefore, the purposeful adaptation of structural disorder brings out special advantages for HK proteins compared to TS proteins. We have demonstrated that TSIs may play vital roles in shaping the molecular adaptation of disordered regions within HK proteins. We also have noticed that HK proteins, mediating a huge number of TSIs, have a greater portion of their interacting interfaces overlapped with the adjacent disordered segment. Moreover, these HK proteins, mediating TSIs, preferably adapt single domain (SD). We have concluded that HK proteins adapt a high degree of structural flexibility to mediate TSIs. Besides, having a SD along with structural flexibility is more economic than maintaining multiple domains with a rigid structure. This assists them in attaining various structural conformations upon binding to their partners, thereby designing an economically optimum molecular system.     

Conservation of polyproline II helices in homologous proteins: implications for structure prediction by model building.

Left-handed polyproline II (PPII) helices commonly occur in globular proteins in segments of 4-8 residues. This paper analyzes the structural conservation of PPII-helices in 3 protein families: serine proteinases, aspartic proteinases, and immunoglobulin constant domains. Calculations of the number of conserved segments based on structural alignment of homologous molecules yielded similar results for the PPII-helices, the alpha-helices, and the beta-strands. The PPII-helices are consistently conserved at the level of 100-80% in the proteins with sequence identity above 20% and RMS deviation of structure alignments below 3.0 A. The most structurally important PPII segments are conserved below this level of sequence identity. These results suggest that the PPII-helices, in addition to the other 2 secondary structure classes, should be identified as part of structurally conserved regions in proteins. This is supported by similar values for the local RMS deviations of the aligned segments for the structural classes of PPII-helices, alpha-helices, and beta-strands. The PPII-helices are shown to participate in supersecondary elements such as PPII-helix/alpha-helix. The conservation of PPII-helices depends on the conservation of a supersecondary element as a whole. PPII-helices also form links, possibly flexible, in the interdomain regions. The role of the PPII-helices in model building by homology is 2-fold; they serve as additional conserved elements in the structure allowing improvement of the accuracy of a model and provide correct chain geometry for modeling of the segments equivalenced to them in a target sequence. The improvement in model building is demonstrated in 2 test studies.     

The pairwise energy content estimated from amino acid composition discriminates between folded and intrinsically unstructured proteins

The structural stability of a protein requires a large number of interresidue interactions. The energetic contribution of these can be approximated by low-resolution force fields extracted from known structures, based on observed amino acid pairing frequencies. The summation of such energies, however, cannot be carried out for proteins whose structure is not known or for intrinsically unstructured proteins. To overcome these limitations, we present a novel method for estimating the total pairwise interaction energy, based on a quadratic form in the amino acid composition of the protein. This approach is validated by the good correlation of the estimated and actual energies of proteins of known structure and by a clear separation of folded and disordered proteins in the energy space it defines. As the novel algorithm has not been trained on unstructured proteins, it substantiates the concept of protein disorder, i.e. that the inability to form a well-defined 3D structure is an intrinsic property of many proteins and protein domains. This property is encoded in their sequence, because their biased amino acid composition does not allow sufficient stabilizing interactions to form. By limiting the calculation to a predefined sequential neighborhood, the algorithm was turned into a position-specific scoring scheme that characterizes the tendency of a given amino acid to fall into an ordered or disordered region. This application we term IUPred and compare its performance with three generally accepted predictors, PONDR VL3H, DISOPRED2 and GlobPlot on a database of disordered proteins.     

The evolution and evolutionary consequences of marginal thermostability in proteins

When we seek to explain the characteristics of living systems in their evolutionary context, we are often interested in understanding how and why certain properties arose through evolution, and how these properties then affected the continuing evolutionary process. This endeavor has been assisted by the use of simple computational models that have properties characteristic of natural living systems but allow simulations over evolutionary timescales with full transparency. We examine a model of the evolution of a gene under selective pressure to code for a protein that exists in a prespecified folded state at a given growth temperature. We observe the emergence of proteins with modest stabilities far below those possible with the model, with a denaturation temperature tracking the simulation temperature, despite the absence of selective pressure for such marginal stability. This demonstrates that neither observations of marginally stable proteins, nor even instances where increased stability interferes with function, provide evidence that marginal stability is an adaptation. Instead the marginal stability is the result of a balance between predominantly destabilizing mutations and selection that shifts depending on effective population size. Even if marginal stability is not an adaptation, the natural tendency of proteins toward marginal stability, and the range of stabilities that occur during evolution, may have significant effect on the evolutionary process.     

Preformed structural elements feature in partner recognition by intrinsically unstructured proteins

Intrinsically unstructured proteins (IUPs) are devoid of extensive structural order but often display signs of local and limited residual structure. To explain their effective functioning, we reasoned that such residual structure can be crucial in their interactions with their structured partner(s) in a way that preformed structural elements presage their final conformational state. To check this assumption, a database of 24 IUPs with known 3D structures in the bound state has been assembled and the distribution of secondary structure elements and backbone torsion angles have been analysed. The high proportion of residues in coil conformation and with phi, psi angles in the disallowed regions of the Ramachandran map compared to the reference set of globular proteins shows that IUPs are not fully ordered even in their bound form. To probe the effect of partner proteins on IUP folding, inherent conformational preferences of IUP sequences have been assessed by secondary structure predictions using the GOR, ALB and PROF algorithms. The accuracy of predicting secondary structure elements of IUPs is similar to that of their partner proteins and is significantly higher than the corresponding values for random sequences. We propose that strong conformational preferences mark regions in IUPs (mostly helices), which correspond to their final structural state, while regions with weak conformational preferences represent flexible linkers between them. In our interpretation, preformed elements could serve as initial contact points, the binding of which facilitates the reeling of the flexible regions onto the template. This finding implies that IUPs draw a functional advantage from preformed structural elements, as they enable their facile, kinetically and energetically less demanding, interaction with their physiological partner.     

Structural segments and residue propensities in protein-RNA interfaces: comparison with protein-protein and protein-DNA complexes

The interface of a protein molecule that is involved in binding another protein, DNA or RNA has been characterized in terms of the number of unique secondary structural segments (SSSs), made up of stretches of helix, strand and non-regular (NR) regions. On average 10-11 segments define the protein interface in protein-protein (PP) and protein-DNA (PD) complexes, while the number is higher (14) for protein-RNA (PR) complexes. While the length of helical segments in PP interaction increases with the interface area, this is not the case in PD and PR complexes. The propensities of residues to occur in the three types of secondary structural elements (SSEs) in the interface relative to the corresponding elements in the protein tertiary structures have been calculated. Arg, Lys, Asn, Tyr, His and Gln are preferred residues in PR complexes; in addition, Ser and Thr are also favoured in PD interfaces.     

Sequence patterns associated with disordered regions in proteins

The relationship between amino acid sequence and intrinsic disorder in proteins is investigated. Two databases, one of disordered proteins and the other of globular proteins, are analyzed and compared in order to extract simple sequence patterns of a few amino acids or amino acid properties that characterize disordered segments. It is found that a number of reliable, nonrandom associations exists. In particular, two types of patterns appear to be recurrent: a proline-rich pattern and a (positively or negatively) charged pattern. These results indicate that local sequence information can determine disordered regions in proteins. The derived patterns provide some insights into the physical reasons for disordered structures. They should also be helpful in improving currently available prediction methods.     

Coils in the membrane core are conserved and functionally important

With the increasing number of available α-helical transmembrane (TM) protein structures, the traditional picture of membrane proteins has been challenged. For example, reentrant regions, which enter and exit the membrane at the same side, and interface helices, which lie parallel with the membrane in the membrane-water interface, are common. Furthermore, TM helices are frequently kinked, and their length and tilt angle vary. Here, we systematically analyze 7% of all residues within the deep membrane core that are in coil state. These coils can be found in TM-helix kinks as major breaks in TM helices and as parts of reentrant regions.Coil residues are significantly more conserved than other residues. Due to the polar character of the coil backbone, they are either buried or located near aqueous channels. Coil residues are frequently found within channels and transporters, where they introduce the flexibility and polarity required for transport across the membrane. Therefore, we believe that coil residues in the membrane core, while constituting a structural anomaly, are essential for the function of proteins.     

Intrinsically disordered regions mediate macromolecular assembly of the Slit diaphragm proteins associated with Nephrotic syndrome

The glomerular filtration barrier (GFB) of the kidney plays an instrumental role in preventing the excretion of large molecules and ensuring the formation of ultra-filtrated urine. Podocytes are essential components of GFB that provide epithelial coverage to the fine glomerular capillaries. Slit-diaphragm (SD) that forms the sole contact between adjacent foot-processes of the podocytes consists of multimeric protein assemblies. SD serves as a molecular sieve and confers size and charge-selective barrier. Nephrin, podocin, TRPC6, and CD2AP are some of the key proteins that constitute the SD. Mutations in these proteins are implicated in nephrotic syndrome and congenital nephropathies which are characterised by heavy proteinuria. The mechanism of how mutations in these proteins predispose to proteinuria is not known. Furthermore, the structural details of proteins that constitute SD are largely unknown. In this study, we built models for nephrin, CD2AP, podocin, and TRPC6 followed by docking and molecular dynamics simulations of the complex of these proteins. We speculate that the interfacial residues of SD proteins form a macromolecular complex through intrinsically disordered regions thereby conferring architectural stability to the SD, which is critical for glomerular permselectivity.     

Context-dependent resistance to proteolysis of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs), also known as intrinsically unstructured proteins (IUPs), lack a well-defined 3D structure in vitro and, in some cases, also in vivo. Here, we discuss the question of proteolytic sensitivity of IDPs, with a view to better explaining their in vivo characteristics. After an initial assessment of the status of IDPs in vivo, we briefly survey the intracellular proteolytic systems. Subsequently, we discuss the evidence for IDPs being inherently sensitive to proteolysis. Such sensitivity would not, however, result in enhanced degradation if the protease-sensitive sites were sequestered. Accordingly, IDP access to and degradation by the proteasome, the major proteolytic complex within eukaryotic cells, are discussed in detail. The emerging picture appears to be that IDPs are inherently sensitive to proteasomal degradation along the lines of the "degradation by default" model. However, available data sets of intracellular protein half-lives suggest that intrinsic disorder does not imply a significantly shorter half-life. We assess the power of available systemic half-life measurements, but also discuss possible mechanisms that could protect IDPs from intracellular degradation. Finally, we discuss the relevance of the proteolytic sensitivity of IDPs to their function and evolution.     

Prediction of natively unfolded regions in protein chains

Analysis showed that the globular or natively unfolded state of a protein can be inferred not only from a lower hydrophobicity or a higher charge, but also from the average environment density (average number of close residues located within a certain distance of a given one) of its residues. A database of 6626 protein structures was used to construct a statistical scale of the average number of close residues in globular structures for the 20 amino acids. The portion of false predictions in distinguishing between 80 globular and 90 natively unfolded proteins was 11% with the new scale and 17% with a hydrophobicity scale. The new scale proved suitable for predicting the folded or unfolded state for native proteins or the natively unfolded regions for protein chains. In comparisons with the available algorithms, the new method yielded the highest portion of true predictions (87 and 77% with averaging over residues and over proteins, respectively).     

A conserved helix-unfolding motif in the naturally unfolded proteins

Among the naturally unfolded proteins there are many polypeptides that retain an extended conformation in the absence of any apparent signal. Using sequence alignment and secondary structure prediction tools, a conserved (LS/SL)(D/E)(D/E)(D/E)X(E/D) motif is uncovered in the vicinity of the N-terminus of their unfolded helices. A comparison of these data with published observations allows one to propose that the (LS/SL)(D/E)(D/E)(D/E)X(E/D) motif is a helix-unfolding signal. Furthermore, the strong similarity between this motif and the STXXDE casein kinase II phosphorylation site suggests a regulatory mechanism for the naturally unfolded proteins within the cell.     

On the relation between residue flexibility and local solvent accessibility in proteins

We investigate the relationship between the flexibility, expressed with B‐factor, and the relative solvent accessibility (RSA) in the context of local, with respect to the sequence, neighborhood and related concepts such as residue depth. We observe that the flexibility of a given residue is strongly influenced by the solvent accessibility of the adjacent neighbors. The mean normalized B‐factor of the exposed residues with two buried neighbors is smaller than that of the buried residues with two exposed neighbors. Inclusion of RSA of the neighboring residues (local RSA) significantly increases correlation with the B‐factor. Correlation between the local RSA and B‐factor is shown to be stronger than the correlation that considers local distance‐ or volume‐based residue depth. We also found that the correlation coefficients between B‐factor and RSA for the 20 amino acids, called flexibility‐exposure correlation index, are strongly correlated with the stability scale that characterizes the average contributions of each amino acid to the folding stability. Our results reveal that the predicted RSA could be used to distinguish between the disordered and ordered residues and that the inclusion of local predicted RSA values helps providing a better contrast between these two types of residues. Prediction models developed based on local actual RSA and local predicted RSA show similar or better results in the context of B‐factor and disorder predictions when compared with several existing approaches. We validate our models using three case studies, which show that this work provides useful clues for deciphering the structure–flexibility–function relation. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Sequence-structure signals of 3D domain swapping in proteins

Three-dimensional domain swapping occurs when two or more identical proteins exchange identical parts of their structure to generate an oligomeric unit. It affects proteins with diverse sequences and structures, and is expected to play important roles in evolution, functional regulation and even conformational diseases. Here, we search for traces of domain swapping in the protein sequence, by means of algorithms that predict the structure and stability of proteins using database-derived potentials. Regions whose sequences are not optimal with regard to the stability of the native structure, or showing marked intrinsic preferences for non-native conformations in absence of tertiary interactions are detected in most domain-swapping proteins. These regions are often located in areas crucial in the swapping process and are likely to influence it on a kinetic or thermodynamic level. In addition, cation-pi interactions are frequently observed to zip up the edges of the interface between intertwined chains or to involve hinge loop residues, thereby modulating stability. We end by proposing a set of mutations altering the swapping propensities, whose experimental characterization would contribute to refine our in silico derived hypotheses.     

The N-terminal domains of SOCS proteins: a conserved region in the disordered N-termini of SOCS4 and 5

Suppressors of cytokine signaling (SOCS) proteins function as negative regulators of cytokine signaling and are involved in fine tuning the immune response. The structure and role of the SH2 domains and C‐terminal SOCS box motifs of the SOCS proteins are well characterized, but the long N‐terminal domains of SOCS4–7 remain poorly understood. Here, we present bioinformatic analyses of the N‐terminal domains of the mammalian SOCS proteins, which indicate that these domains of SOCS4, 5, 6, and 7 are largely disordered. We have also identified a conserved region of about 70 residues in the N‐terminal domains of SOCS4 and 5 that is predicted to be more ordered than the surrounding sequence. The conservation of this region can be traced as far back as lower vertebrates. As conserved regions with increased structural propensity that are located within long disordered regions often contain molecular recognition motifs, we expressed the N‐terminal conserved region of mouse SOCS4 for further analysis. This region, mSOCS4_86–155, has been characterized by circular dichroism and nuclear magnetic resonance spectroscopy, both of which indicate that it is predominantly unstructured in aqueous solution, although it becomes helical in the presence of trifluoroethanol. The high degree of sequence conservation of this region across different species and between SOCS4 and SOCS5 nonetheless implies that it has an important functional role, and presumably this region adopts a more ordered conformation in complex with its partners. The recombinant protein will be a valuable tool in identifying these partners and defining the structures of these complexes. Proteins 2011. © 2012 Wiley Periodicals, Inc.