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.     

Molecular Recognition Features in Zika Virus Proteome

Viruses have compact genomes that encode limited number of proteins in comparison to other biological entities. Interestingly, viral proteins have shown natural abundance of either completely disordered proteins that are recognized as intrinsically disorder proteins (IDPs) or partially disordered segments known as intrinsically disordered protein regions (IDPRs). IDPRs are involved in interactions with multiple binding partners to accomplish signaling, regulation, and control functions in cells. Tuning of IDPs and IDPRs are mediated through post-translational modification and alternative splicing. Often, the interactions of IDPRs with their binding protein partner(s) lead to transition from the state of disorder to ordered form. Such interaction-prone protein IDPRs are identified as molecular recognition features (MoRFs). Molecular recognition is an important initial step for the biomolecular interactions and their functional proceedings. Although previous studies have established occurrence of the IDPRs in Zika virus proteome, which provide the functional diversity and structural plasticity to viral proteins, the MoRF analysis has not been performed as of yet. Many computational methods have been developed for the identification of the MoRFs in protein sequences including ANCHOR, MoRFpred, DISOPRED3, and MoRFchibi_web server. In the current study, we have investigated the presence of MoRF regions in structural and non-structural proteins of Zika virus using an aforementioned set of computational techniques. Furthermore, we have experimentally validated the intrinsic disorderness of NS2B cofactor region of NS2B–NS3 protease. NS2B has one of the longest MoRF regions in Zika virus proteome. In future, this study may provide valuable information while investigating the virus host protein interaction networks.     

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.     

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).     

Does Lack of Secondary Structure Imply Intrinsic Disorder in Proteins? A Sequence Analysis

Intrinsically disordered proteins (IDPs)/protein regions (IDPRs) lack unique three-dimensional structure at the level of secondary and/or tertiary structure and are represented as an ensemble of interchanging conformations. To investigate the role of presence/absence of secondary structures in promoting intrinsic disorder in proteins, a comparative sequence analysis of IDPs, IDPRs and proteins with minimal secondary structures (less than 5%) is required. A sequence analysis reveals proteins with minimal secondary structure content have high mean net positive charge, low mean net hydrophobicity and low sequence complexity. Interestingly, analysis of the relative local electrostatic interactions reveal that an increase in the relative repulsive interactions between amino acids separated by three or four residues lead to either loss of secondary structure or intrinsic disorder. IDPRs show increase in both local negative-negative and positive-positive repulsive interactions. While IDPs show a marked increase in the local negative-negative interactions, proteins with minimal secondary structure depict an increase in the local positive-positive interactions. IDPs and IDPRs are enriched in D, E and Q residues, while proteins with minimal secondary structure are depleted of these residues. Proteins with minimal secondary structures have higher content of G and C, while IDPs and IDPRs are depleted of these residues. These results confirm that proteins with minimal secondary structure have a distinctly different propensity for charge, hydrophobicity, specific amino acids and local electrostatic interactions as compared to IDPs/IDPRs. Thus we conclude that lack of secondary structure may be a necessary but not a sufficient condition for intrinsic disorder in proteins.     

Identification of Atg3 as an intrinsically disordered polypeptide yields insights into the molecular dynamics of autophagy-related proteins in yeast

The mechanism of autophagy relies on complex cell signaling and regulatory processes. Each cell contains many proteins that lack a rigid 3-dimensional structure under physiological conditions. These dynamic proteins, called intrinsically disordered proteins (IDPs) and protein regions (IDPRs), are predominantly involved in cell signaling and regulation. Yet, very little is known about their presence among proteins of the core autophagy machinery. In this work, we characterized the autophagy protein Atg3 from yeast and human along with 2 variants to show that Atg3 is an IDPRs-containing protein and that disorder/order predicted for these proteins from their amino acid sequence corresponds to their experimental characteristics. Based on this consensus, we applied the same prediction methods to all known Atg proteins from Saccharomyces cerevisiae. The data presented here provide an insight into the structural dynamics of each Atg protein. They also show that intrinsic disorder at various levels has to be taken into consideration for about half of the Atg proteins. This work should become a useful tool that will facilitate and encourage exploration of protein intrinsic disorder in autophagy.     

Identification of Atg3 as an intrinsically disordered polypeptide yields insights into the molecular dynamics of autophagy-related proteins in yeast

The mechanism of autophagy relies on complex cell signaling and regulatory processes. Each cell contains many proteins that lack a rigid 3-dimensional structure under physiological conditions. These dynamic proteins, called intrinsically disordered proteins (IDPs) and protein regions (IDPRs), are predominantly involved in cell signaling and regulation. Yet, very little is known about their presence among proteins of the core autophagy machinery. In this work, we characterized the autophagy protein Atg3 from yeast and human along with 2 variants to show that Atg3 is an IDPRs-containing protein and that disorder/order predicted for these proteins from their amino acid sequence corresponds to their experimental characteristics. Based on this consensus, we applied the same prediction methods to all known Atg proteins from Saccharomyces cerevisiae. The data presented here provide an insight into the structural dynamics of each Atg protein. They also show that intrinsic disorder at various levels has to be taken into consideration for about half of the Atg proteins. This work should become a useful tool that will facilitate and encourage exploration of protein intrinsic disorder in autophagy.     

Resolving the ambiguity: Making sense of intrinsic disorder when PDB structures disagree

Missing regions in X‐ray crystal structures in the Protein Data Bank (PDB) have played a foundational role in the study of intrinsically disordered protein regions (IDPRs), especially in the development of in silico predictors of intrinsic disorder. However, a missing region is only a weak indication of intrinsic disorder, and this uncertainty is compounded by the presence of ambiguous regions, where more than one structure of the same protein sequence “disagrees” in terms of the presence or absence of missing residues. The question is this: are these ambiguous regions intrinsically disordered, or are they the result of static disorder that arises from experimental conditions, ensembles of structures, or domain wobbling? A novel way of looking at ambiguous regions in terms of the pattern between multiple PDB structures has been demonstrated. It was found that the propensity for intrinsic disorder increases as the level of ambiguity decreases. However, it is also shown that ambiguity is more likely to occur as the protein region is placed within different environmental conditions, and even the most ambiguous regions as a set display compositional bias that suggests flexibility. The results suggested that ambiguity is a natural result for many IDPRs crystallized under different conditions and that static disorder and wobbling domains are relatively rare. Instead, it is more likely that ambiguity arises because many of these regions were conditionally or partially disordered.     

On the Potential of Machine Learning to Examine the Relationship Between Sequence,Structure, Dynamics and Function of Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) constitute a broad set of proteins with few uniting and many diverging properties. IDPs—and intrinsically disordered regions (IDRs) interspersed between folded domains—are generally characterized as having no persistent tertiary structure; instead they interconvert between a large number of different and often expanded structures. IDPs and IDRs are involved in an enormously wide range of biological functions and reveal novel mechanisms of interactions, and while they defy the common structure-function paradigm of folded proteins, their structural preferences and dynamics are important for their function. We here discuss open questions in the field of IDPs and IDRs, focusing on areas where machine learning and other computational methods play a role. We discuss computational methods aimed to predict transiently formed local and long-range structure, including methods for integrative structural biology. We discuss the many different ways in which IDPs and IDRs can bind to other molecules, both via short linear motifs, as well as in the formation of larger dynamic complexes such as biomolecular condensates. We discuss how experiments are providing insight into such complexes and may enable more accurate predictions. Finally, we discuss the role of IDPs in disease and how new methods are needed to interpret the mechanistic effects of genomic variants in IDPs.     

Utilization of protein intrinsic disorder knowledge in structural proteomics

Intrinsically disordered proteins (IDPs) and proteins with long disordered regions are highly abundant in various proteomes. Despite their lack of well-defined ordered structure, these proteins and regions are frequently involved in crucial biological processes. Although in recent years these proteins have attracted the attention of many researchers, IDPs represent a significant challenge for structural characterization since these proteins can impact many of the processes in the structure determination pipeline. Here we investigate the effects of IDPs on the structure determination process and the utility of disorder prediction in selecting and improving proteins for structural characterization. Examination of the extent of intrinsic disorder in existing crystal structures found that relatively few protein crystal structures contain extensive regions of intrinsic disorder. Although intrinsic disorder is not the only cause of crystallization failures and many structured proteins cannot be crystallized, filtering out highly disordered proteins from structure-determination target lists is still likely to be cost effective. Therefore it is desirable to avoid highly disordered proteins from structure-determination target lists and we show that disorder prediction can be applied effectively to enrich structure determination pipelines with proteins more likely to yield crystal structures. For structural investigation of specific proteins, disorder prediction can be used to improve targets for structure determination. Finally, a framework for considering intrinsic disorder in the structure determination pipeline is proposed.     

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.     

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.     

Targeting intrinsically disordered proteins in neurodegenerative and protein dysfunction diseases: another illustration of the D2 concept

Many biologically active proteins, which are usually called intrinsically disordered or natively unfolded proteins, lack stable tertiary and/or secondary structure under physiological conditions in vitro. Their functions complement the functional repertoire of ordered proteins, with intrinsically disordered proteins (IDPs) often being involved in regulation, signaling and control. Their amino acid sequences and compositions are very different from those of ordered proteins, making reliable identification of IDPs possible at the proteome level. IDPs are highly abundant in various human diseases, including neurodegeneration and other protein dysfunction maladies and, therefore, represent attractive novel drug targets. Some of the aspects of IDPs, as well as their roles in neurodegeneration and protein dysfunction diseases, are discussed in this article, together with the peculiarities of IDPs as potential drug targets.     

固有无序蛋白研究进展及其对细胞胁迫耐受性的影响

固有无序蛋白(intrinsically disordered proteins,IDPs)是指在生理条件下缺乏有序稳定的高级结构,整体或局部不折叠,但能够参与多种生物学过程、行使特定生物学功能的一类蛋白质.固有无序蛋白决定了其不同于经典蛋白质"序列-结构-功能"的功能范式,丰富了蛋白质"结构-功能"的多样性.固有无序...     

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.     

固有无序蛋白质及逆境胁迫下的分子功能

固有无序蛋白质是一类在生理条件下缺乏稳定三维结构而具有正常功能,参与信号转导、转录调控、胁迫应答等多种生物学过程的蛋白质.植物中许多逆境响应蛋白是固有无序蛋白质,通过其结构无序或部分无序区域在蛋白质 蛋白质、蛋白质 膜脂、蛋白质 核酸的互作中发挥重要作用.本文主要对固有无序蛋白质的类别、氨基酸组成和结构特点以及在逆境胁迫下其稳定细胞膜、保护核酸和蛋白质、调控基因表达等分子功能进行综述,以拓展对逆境胁迫下蛋白质作用分子机制的认识.     

Resilience of death: intrinsic disorder in proteins involved in the programmed cell death

It is recognized now that intrinsically disordered proteins (IDPs), which do not have unique 3D structures as a whole or in noticeable parts, constitute a significant fraction of any given proteome. IDPs are characterized by an astonishing structural and functional diversity that defines their ability to be universal regulators of various cellular pathways. Programmed cell death (PCD) is one of the most intricate cellular processes where the cell uses specialized cellular machinery and intracellular programs to kill itself. This cell-suicide mechanism enables metazoans to control cell numbers and to eliminate cells that threaten the animal''s survival. PCD includes several specific modules, such as apoptosis, autophagy, and programmed necrosis (necroptosis). These modules are not only tightly regulated but also intimately interconnected and are jointly controlled via a complex set of protein–protein interactions. To understand the role of the intrinsic disorder in controlling and regulating the PCD, several large sets of PCD-related proteins across 28 species were analyzed using a wide array of modern bioinformatics tools. This study indicates that the intrinsic disorder phenomenon has to be taken into consideration to generate a complete picture of the interconnected processes, pathways, and modules that determine the essence of the PCD. We demonstrate that proteins involved in regulation and execution of PCD possess substantial amount of intrinsic disorder. We annotate functional roles of disorder across and within apoptosis, autophagy, and necroptosis processes. Disordered regions are shown to be implemented in a number of crucial functions, such as protein–protein interactions, interactions with other partners including nucleic acids and other ligands, are enriched in post-translational modification sites, and are characterized by specific evolutionary patterns. We mapped the disorder into an integrated network of PCD pathways and into the interactomes of selected proteins that are involved in the p53-mediated apoptotic signaling pathway.     

Close encounters of the third kind: disordered domains and the interactions of proteins

Protein–protein interactions are thought to be mediated by domains, which are autonomous folding units of proteins. Recently, a second type of interaction has been suggested, mediated by short segments termed linear motifs, which are related to recognition elements of intrinsically disordered regions. Here, we propose a third kind of protein–protein recognition mechanism, mediated by disordered regions longer than 20–30 residues. Bioinformatics predictions and well‐characterized examples, such as the kinase‐inhibitory domain of Cdk inhibitors and the Wiskott–Aldrich syndrome protein (WASP)‐homology domain 2 of actin‐binding proteins, show that these disordered regions conform to the definition of domains rather than motifs, i.e., they represent functional, evolutionary, and structural units. Their functions are distinct from those of short motifs and ordered domains, and establish a third kind of interaction principle. With these points, we argue that these long disordered regions should be recognized as a distinct class of biologically functional protein domains.     

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.     

Smoothing molecular interactions: the "kinetic buffer" effect of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) widely participate in molecular recognition and signaling processes in cells by interacting with other molecules. Compared with ordered proteins, IDPs usually possess stronger intermolecular interactions in binding. As a result, the interface structure of IDPs in complexes is distinct from that of ordered-protein complexes, and this difference may have essential effect on the response to various perturbations in a cell. In this study, we examined the perturbations of intermolecular interactions and temperature on the coupled folding and binding processes of pKID to KIX domains by performing molecular dynamics simulations. By comparing a series of virtual pKID systems with various degree of disorder, we found that the complex stability and the binding kinetics of the disordered systems were less sensitive to the perturbations than the ordered systems. The origin of the lower response sensitivity of IDPs was attributed to their higher flexibility in the complex interface, which was further supported by an analysis on protein complex structures. On the basis of our simulations and results from the literature, we speculate IDPs may not only interact with their biological partners with high specificity and low affinity but also may be resistant to the perturbations in the environment and transmit signals fast and smooth. We proposed to name it the "kinetic buffer" effect.