Advantages of proteins being disordered

The past decade has witnessed great advances in our understanding of protein structure‐function relationships in terms of the ubiquitous existence of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs). The structural disorder of IDPs/IDRs enables them to play essential functions that are complementary to those of ordered proteins. In addition, IDPs/IDRs are persistent in evolution. Therefore, they are expected to possess some advantages over ordered proteins. In this review, we summarize and survey nine possible advantages of IDPs/IDRs: economizing genome/protein resources, overcoming steric restrictions in binding, achieving high specificity with low affinity, increasing binding rate, facilitating posttranslational modifications, enabling flexible linkers, preventing aggregation, providing resistance to non‐native conditions, and allowing compatibility with more available sequences. Some potential advantages of IDPs/IDRs are not well understood and require both experimental and theoretical approaches to decipher. The connection with protein design is also briefly discussed.     

Does Intrinsic Disorder in Proteins Favor Their Interaction with Lipids?

Intrinsically disordered proteins (IDPs) are implicated in a range of human diseases, some of which are associated with the ability to bind to lipids. Although the presence of solvent‐exposed hydrophobic regions in IDPs should favor their interactions with low‐molecular‐weight hydrophobic/amphiphilic compounds, this hypothesis has not been systematically explored as of yet. In this study, the analysis of the DisProt database with regard to the presence of lipid‐binding IDPs (LBIDPs) reveals that they comprise, at least, 15% of DisProt entries. LBIDPs are classified into four groups by ligand type, functional categories, domain structure, and conformational state. 57% of LBIDPs are classified as ordered according to the CH‐CDF analysis, and 70% of LBIDPs possess lengths of disordered regions below 50%. To investigate the lipid‐binding properties of IDPs for which lipid binding is not reported, three proteins from different conformational groups are rationally selected. They all are shown to bind linoleic (LA) and oleic (OA) acids with capacities ranging from 9 to 34 LA/OA molecules per protein molecule. The association with LA/OA causes the formation of high‐molecular‐weight lipid–protein complexes. These findings suggest that lipid binding is common among IDPs, which can favor their involvement in lipid metabolism.     

The disordered PCI‐binding human proteins CSNAP and DSS1 have diverged in structure and function

Intrinsically disordered proteins (IDPs) regularly constitute components of larger protein assemblies contributing to architectural stability. Two small, highly acidic IDPs have been linked to the so‐called PCI complexes carrying PCI‐domain subunits, including the proteasome lid and the COP9 signalosome. These two IDPs, DSS1 and CSNAP, have been proposed to have similar structural propensities and functions, but they display differences in their interactions and interactome sizes. Here we characterized the structural properties of human DSS1 and CSNAP at the residue level using NMR spectroscopy and probed their propensities to bind ubiquitin. We find that distinct structural features present in DSS1 are completely absent in CSNAP, and vice versa, with lack of relevant ubiquitin binding to CSNAP, suggesting the two proteins to have diverged in both structure and function. Our work additionally highlights that different local features of seemingly similar IDPs, even subtle sequence variance, may endow them with different functional traits. Such traits may underlie their potential to engage in multiple interactions thereby impacting their interactome sizes.     

Differential dehydration effects on globular proteins and intrinsically disordered proteins during film formation

Globular proteins composed of different secondary structures and fold types were examined by synchrotron radiation circular dichroism spectroscopy to determine the effects of dehydration on their secondary structures. They exhibited only minor changes upon removal of bulk water during film formation, contrary to previously reported studies of proteins dehydrated by lyophilization (where substantial loss of helical structure and gain in sheet structure was detected). This near lack of conformational change observed for globular proteins contrasts with intrinsically disordered proteins (IDPs) dried in the same manner: the IDPs, which have almost completely unordered structures in solution, exhibited increased amounts of regular (mostly helical) secondary structures when dehydrated, suggesting formation of new intra‐protein hydrogen bonds replacing solvent‐protein hydrogen bonds, in a process which may mimic interactions that occur when IDPs bind to partner molecules. This study has thus shown that the secondary structures of globular and intrinsically disordered proteins behave very differently upon dehydration, and that films are a potentially useful format for examining dehydrated soluble proteins and assessing IDPs structures.     

Obtaining highly purified intrinsically disordered protein by boiling lysis and single step ion exchange

Intrinsically disordered proteins (IDPs) is a term used to describe proteins that do not have a well-defined tertiary structure. IDPs have many roles such as in cell cycle control (p53), neuronal signal transmission (myelin basic protein), and protein stability (dehydrins). Producing recombinant IDPs in bacteria for nuclear magnetic resonance (NMR) studies is problematic because the lack of stable tertiary structure makes them excellent substrates for bacterial proteases, which will cause loss in yield. We have developed a two-step method to produce the grape dehydrin K₂ and YSK₂ using Escherichia coli. Dehydrins are expressed by certain plants in response to dehydration, increased salinity, or low temperatures. Purification of 10 mg/L (K₂) and 15 mg/L (YSK₂) was performed by boiling bacterial pellets to lyse the cells, remove most of the contaminating proteins, and denature bacterial proteases. This resulted in protein purity comparable to that produced by sonication and nickel affinity chromatography. Boiling was followed by cation exchange chromatography to remove the remaining trace contaminants. The sample was shown to be more than 95% pure by reversed-phase high-performance liquid chromatography. The method presented here can easily be adapted to the purification of other IDPs and heat-stable proteins without requiring multiple chromatography steps or the use of protease inhibitors.     

Small Molecule Sequestration of the Intrinsically Disordered Protein,p27Kip1, Within Soluble Oligomers

Proteins that exhibit intrinsically disordered regions (IDRs) are prevalent in the human proteome and perform diverse biological functions, including signaling and regulation. Due to these important roles, misregulation of intrinsically disordered proteins (IDPs) is associated with myriad human diseases, including neurodegeneration and cancer. The inherent flexibility of IDPs limits the applicability of the traditional structure-based drug design paradigm; therefore, IDPs have long been considered “undruggable”. Using NMR spectroscopy and other methods, we previously discovered small, drug-like molecules that bind specifically, albeit weakly, to dynamic clusters of aromatic residues within p27^Kip1 (p27), an archetypal disordered protein involved in cell cycle regulation. Here, using synthetic chemistry, NMR spectroscopy and other biophysical methods, we discovered elaborated analogs of our previously reported molecules with 30-fold increased affinity for p27 (apparent K_d = 57 ± 19 μM). Strikingly, using analytical ultracentrifugation methods, we showed that the highest affinity compounds caused p27 to form soluble, disordered oligomers. Based on these observations, we propose that sequestration within soluble oligomers may represent a general strategy for therapeutically targeting disease-associated IDPs in the future.     

Modulation of Correlated Segment Fluctuations in IDPs upon Complex Formation as an Allosteric Regulatory Mechanism

Molecular recognition of and by intrinsically disordered proteins (IDPs) is an intriguing and still largely elusive phenomenon. Typically, protein recognition involving IDPs requires either folding upon binding or, alternatively, the formation of “fuzzy complexes.” Here we show via correlation analyses of paramagnetic relaxation enhancement data unprecedented and striking alterations of the concerted fluctuations within the conformational ensemble of IDPs upon ligand binding. We study the binding of α-synuclein to calmodulin, a ubiquitous calcium-binding protein, and the binding of the extracellular matrix IDP osteopontin to heparin, a mimic of the extracellular matrix ligand hyaluronic acid. In both cases, binding leads to reduction of correlated long-range motions in these two IDPs and thus indicates a loosening of structural compaction upon binding. Most importantly, however, the simultaneous presence of correlated and anti-correlated fluctuations in IDPs suggests the prevalence of “energetic frustration” and provides an explanation for the puzzling observation of disordered allostery in IDPs.     

Microtubule-associated protein (MAP) 4 interacts with microtubules in an intrinsically disordered manner

We previously used nuclear magnetic resonance (NMR) to analyze the structure of a synthetic tricosapeptide corresponding to an active site of microtubule-associated protein 4 (MAP4). To further the structural analysis, we have constructed a minimal active domain fragment of MAP4, encompassing the entire active site, and obtained its NMR spectra. The secondary structure prediction using partially assigned NMR data suggested that the fragment is largely unfolded. Two other independent techniques also demonstrated its unfolded nature, indicating that MAP4 belongs to the class of intrinsically disordered proteins (IDPs). The NMR spectra of the fragment-microtubule mixture revealed that the fragment binds to the microtubule using multiple binding sites, apparently contradicting our previous quantitative studies. Given that MAP4 is intrinsically disordered, we propose a mechanism in which any one of the binding sites is active at a time, which is one of the typical interaction mechanisms proposed for IDPs.     

Self-assembling systems comprising intrinsically disordered protein polymers like elastin-like recombinamers

Despite lacking cooperatively folded structures under native conditions, numerous intrinsically disordered proteins (IDPs) nevertheless have great functional importance. These IDPs are hybrids containing both ordered and intrinsically disordered protein regions (IDPRs), the structure of which is highly flexible in this unfolded state. The conformational flexibility of these disordered systems favors transitions between disordered and ordered states triggered by intrinsic and extrinsic factors, folding into different dynamic molecular assemblies to enable proper protein functions. Indeed, prokaryotic enzymes present less disorder than eukaryotic enzymes, thus showing that this disorder is related to functional and structural complexity. Protein-based polymers that mimic these IDPs include the so-called elastin-like polypeptides (ELPs), which are inspired by the composition of natural elastin. Elastin-like recombinamers (ELRs) are ELPs produced using recombinant techniques and which can therefore be tailored for a specific application. One of the most widely used and studied characteristic structures in this field is the pentapeptide (VPGXG)_n. The structural disorder in ELRs probably arises due to the high content of proline and glycine in the ELR backbone, because both these amino acids help to keep the polypeptide structure of elastomers disordered and hydrated. Moreover, the recombinant nature of these systems means that different sequences can be designed, including bioactive domains, to obtain specific structures for each application. Some of these structures, along with their applications as IDPs that self-assemble into functional vesicles or micelles from diblock copolymer ELRs, will be studied in the following sections. The incorporation of additional order- and disorder-promoting peptide/protein domains, such as α-helical coils or β-strands, in the ELR sequence, and their influence on self-assembly, will also be reviewed. In addition, chemically cross-linked systems with controllable order–disorder balance, and their role in biomineralization, will be discussed. Finally, we will review different multivalent IDPs-based coatings and films for different biomedical applications, such as spatially controlled cell adhesion, osseointegration, or biomaterial-associated infection (BAI).     

Nucleocytoplasmic transport of intrinsically disordered proteins studied by high‐speed super‐resolution microscopy

Both natively folded and intrinsically disordered proteins (IDPs) destined for the nucleus need to transport through the nuclear pore complexes (NPCs) in eukaryotic cells. NPCs allow for passive diffusion of small folded proteins while barricading large ones, unless they are facilitated by nuclear transport receptors. However, whether nucleocytoplasmic transport of IDPs would follow these rules remains unknown. By using a high‐speed super‐resolution fluorescence microscopy, we have measured transport kinetics and 3D spatial locations of transport routes through native NPCs for various IDPs. Our data revealed that the rules executed for folded proteins are not well followed by the IDPs. Instead, both large and small IDPs can passively diffuse through the NPCs. Furthermore, their diffusion efficiencies and routes are differentiated by their content ratio of charged (Ch) and hydrophobic (Hy) amino acids. A Ch/Hy‐ratio mechanism was finally suggested for nucleocytoplasmic transport of IDPs.     

Features of molecular recognition of intrinsically disordered proteins via coupled folding and binding

The sequence–structure–function paradigm of proteins has been revolutionized by the discovery of intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs). In contrast to traditional ordered proteins, IDPs/IDRs are unstructured under physiological conditions. The absence of well‐defined three‐dimensional structures in the free state of IDPs/IDRs is fundamental to their function. Folding upon binding is an important mode of molecular recognition for IDPs/IDRs. While great efforts have been devoted to investigating the complex structures and binding kinetics and affinities, our knowledge on the binding mechanisms of IDPs/IDRs remains very limited. Here, we review recent advances on the binding mechanisms of IDPs/IDRs. The structures and kinetic parameters of IDPs/IDRs can vary greatly, and the binding mechanisms can be highly dependent on the structural properties of IDPs/IDRs. IDPs/IDRs can employ various combinations of conformational selection and induced fit in a binding process, which can be templated by the target and/or encoded by the IDP/IDR. Further studies should provide deeper insights into the molecular recognition of IDPs/IDRs and enable the rational design of IDP/IDR binding mechanisms in the future.     

Intrinsically disordered proteins: regulation and disease

Intrinsically disordered proteins (IDPs) are enriched in signaling and regulatory functions because disordered segments permit interaction with several proteins and hence the re-use of the same protein in multiple pathways. Understanding IDP regulation is important because altered expression of IDPs is associated with many diseases. Recent studies show that IDPs are tightly regulated and that dosage-sensitive genes encode proteins with disordered segments. The tight regulation of IDPs may contribute to signaling fidelity by ensuring that IDPs are available in appropriate amounts and not present longer than needed. The altered availability of IDPs may result in sequestration of proteins through non-functional interactions involving disordered segments (i.e., molecular titration), thereby causing an imbalance in signaling pathways. We discuss the regulation of IDPs, address implications for signaling, disease and drug development, and outline directions for future research.     

Binding cavities and druggability of intrinsically disordered proteins

To assess the potential of intrinsically disordered proteins (IDPs) as drug design targets, we have analyzed the ligand-binding cavities of two datasets of IDPs (containing 37 and 16 entries, respectively) and compared their properties with those of conventional ordered (folded) proteins. IDPs were predicted to possess more binding cavity than ordered proteins at similar length, supporting the proposed advantage of IDPs economizing genome and protein resources. The cavity number has a wide distribution within each conformation ensemble for IDPs. The geometries of the cavities of IDPs differ from the cavities of ordered proteins, for example, the cavities of IDPs have larger surface areas and volumes, and are more likely to be composed of a single segment. The druggability of the cavities was examined, and the average druggable probability is estimated to be 9% for IDPs, which is almost twice that for ordered proteins (5%). Some IDPs with druggable cavities that are associated with diseases are listed. The optimism versus obstacles for drug design for IDPs is also briefly discussed.     

Evolutionarily Conserved Network Properties of Intrinsically Disordered Proteins

BackgroundIntrinsically disordered proteins (IDPs) lack a stable tertiary structure in isolation. Remarkably, however, a substantial portion of IDPs undergo disorder-to-order transitions upon binding to their cognate partners. Structural flexibility and binding plasticity enable IDPs to interact with a broad range of partners. However, the broader network properties that could provide additional insights into the functional role of IDPs are not known.ResultsHere, we report the first comprehensive survey of network properties of IDP-induced sub-networks in multiple species from yeast to human. Our results show that IDPs exhibit greater-than-expected modularity and are connected to the rest of the protein interaction network (PIN) via proteins that exhibit the highest betweenness centrality and connect to fewer-than-expected IDP communities, suggesting that they form critical communication links from IDP modules to the rest of the PIN. Moreover, we found that IDPs are enriched at the top level of regulatory hierarchy.ConclusionOverall, our analyses reveal coherent and remarkably conserved IDP-centric network properties, namely, modularity in IDP-induced network and a layer of critical nodes connecting IDPs with the rest of the PIN.     

Cancer-Associated Mutations Perturb the Disordered Ensemble and Interactions of the Intrinsically Disordered p53 Transactivation Domain

Intrinsically disordered proteins (IDPs) are key components of regulatory networks that control crucial aspects of cell decision making. The intrinsically disordered transactivation domain (TAD) of tumor suppressor p53 mediates its interactions with multiple regulatory pathways to control the p53 homeostasis during the cellular response to genotoxic stress. Many cancer-associated mutations have been discovered in p53-TAD, but their structural and functional consequences are poorly understood. Here, by combining atomistic simulations, NMR spectroscopy, and binding assays, we demonstrate that cancer-associated mutations can significantly perturb the balance of p53 interactions with key activation and degradation regulators. Importantly, the four mutations studied in this work do not all directly disrupt the known interaction interfaces. Instead, at least three of these mutations likely modulate the disordered state of p53-TAD to perturb its interactions with regulators. Specifically, NMR and simulation analysis together suggest that these mutations can modulate the level of conformational expansion as well as rigidity of the disordered state. Our work suggests that the disordered conformational ensemble of p53-TAD can serve as a central conduit in regulating the response to various cellular stimuli at the protein–protein interaction level. Understanding how the disordered state of IDPs may be modulated by regulatory signals and/or disease associated perturbations will be essential in the studies on the role of IDPs in biology and diseases.     

Cold stability of intrinsically disordered proteins

Contrary to globular proteins, intrinsically disordered proteins (IDPs) lack a folded structure and they do not lose solubility at elevated temperatures. Although this should also be true at low temperatures, cold stability of IDPs has not been addressed in any scientific work so far. As direct characterization of cold-denaturation is difficult, we approached the problem through a freezing-induced loss-of-function model of globular-disordered functional protein pairs (m-calpain-calpastatin, tubulin-Map2c, Hsp90-ERD14). Our results affirm that in contrast with globular proteins IDPs are resistant to cold treatment. The theoretical and functional aspects of this observation are discussed.