Binding Of Intrinsically Disordered Proteins Research Articles

A New Tool to Study the Binding Behavior of Intrinsically Disordered Proteins.

Understanding the binding behavior and conformational dynamics of intrinsically disordered proteins (IDPs) is crucial for unraveling their regulatory roles in biological processes. However, their lack of stable 3D structures poses challenges for analysis. To address this, we propose an algorithm that explores IDP binding behavior with protein complexes by extracting topological and geometric features from the protein surface model. Our algorithm identifies a geometrically favorable binding pose for the IDP and plans a feasible trajectory to evaluate its transition to the docking position. We focus on IDPs from Homo sapiens and Mus-musculus, investigating their interaction with the Plasmodium falciparum (PF) pathogen associated with malaria-related deaths. We compare our algorithm with HawkDock and HDOCK docking tools for quantitative (computation time) and qualitative (binding affinity) measures. Our results indicated that our method outperformed the compared methods in computation performance and binding affinity in experimental conformations.

Mutational scan inferred binding energetics and structure in intrinsically disordered protein CcdA.

Unlike globular proteins, mutational effects on the function of Intrinsically Disordered Proteins (IDPs) are not well-studied. Deep Mutational Scanning of a yeast surface displayed mutant library yields insights into sequence-function relationships in the CcdA IDP. The approach enables facile prediction of interface residues and local structural signatures of the bound conformation. In contrast to previous titration-based approaches which use a number of ligand concentrations, we show that use of a single rationally chosen ligand concentration can provide quantitative estimates of relative binding constants for large numbers of protein variants. This is because the extended interface of IDP ensures that energetic effects of point mutations are spread over a much smaller range than for globular proteins. Our data also provides insights into the much-debated role of helicity and disorder in partner binding of IDPs. Based on this exhaustive mutational sensitivity dataset, a rudimentary model was developed in an attempt to predict mutational effects on binding affinity of IDPs that form alpha-helical structures upon binding.

Prediction of protein-protein interaction sites in intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) participate in many biological processes by interacting with other proteins, including the regulation of transcription, translation, and the cell cycle. With the increasing amount of disorder sequence data available, it is thus crucial to identify the IDP binding sites for functional annotation of these proteins. Over the decades, many computational approaches have been developed to predict protein-protein binding sites of IDP (IDP-PPIS) based on protein sequence information. Moreover, there are new IDP-PPIS predictors developed every year with the rapid development of artificial intelligence. It is thus necessary to provide an up-to-date overview of these methods in this field. In this paper, we collected 30 representative predictors published recently and summarized the databases, features and algorithms. We described the procedure how the features were generated based on public data and used for the prediction of IDP-PPIS, along with the methods to generate the feature representations. All the predictors were divided into three categories: scoring functions, machine learning-based prediction, and consensus approaches. For each category, we described the details of algorithms and their performances. Hopefully, our manuscript will not only provide a full picture of the status quo of IDP binding prediction, but also a guide for selecting different methods. More importantly, it will shed light on the inspirations for future development trends and principles.

Electronic Polarization at the Interface between the p53 Transactivation Domain and Two Binding Partners.

Intrinsically disordered proteins (IDPs) are an abundant class of highly charged proteins that participate in numerous crucial biological processes, often in regulatory roles. IDPs do not have one major free energy minimum with a dominant structure, instead existing as conformational ensembles of multiple semistable conformations. p53 is a prototypical protein with disordered regions and binds to many structurally diverse partners, making it a useful model for exploring the role of electrostatic interactions at IDP binding interfaces. In this study, we used the Drude-2019 force field to simulate the p53 transactivation domain with two protein partners to probe the role of electrostatic interactions in IDP protein-protein interactions. We found that the Drude-2019 polarizable force field reasonably reproduced experimental chemical shifts of the p53 transactivation domain (TAD) in one complex for which these data are available. We also found that the proteins in these complexes displayed dipole response at specific residues of each protein and that residues primarily involved in binding showed a large percent change in dipole moment between the unbound and complexed states. Probing the role of electrostatic interactions in IDP binding can allow us greater fundamental understanding of these interactions and may help with targeting p53 or its partners for drug design.

Portability of Small Molecule Binding Sites in Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) lack stable secondary and tertiary structure. Because they are associated with a range of diseases, IDPs are desirable therapeutic targets and have been targeted by small molecules. The transcription factor c‐Myc (Myc) is an IDP that is implicated in over 70% of cancers. Previously, we have shown that several small molecules can bind to short, contiguous sequences on Myc. These sequences remain unstructured when binding to small molecules, potentially indicating only a limited dependence on residues outside of the contiguous binding residues. This mode of binding differs from that common in structured proteins where contact residues are often noncontiguous and scaffolded by extensive protein structure. Here we test the portability of a small molecule‐IDP interaction implied by its dependence on only a short, linear sequence. We compare the binding of a small molecule to its specific recognition sequence in a native Myc context, in a minimalist peptide context, and by porting the binding sequence into the context of a different IDP. An ability of short, disordered sequences to bind small molecules in a context agnostic manner would have implications for the prediction and discovery of IDP binding sites broadly.

The role of membranes in function and dysfunction of intrinsically disordered amyloidogenic proteins.

Membrane-protein interactions play a major role in human physiology as well as in diseases pathology. Interaction of a protein with the membrane was previously thought to be dependent on well-defined three-dimensional structure of the protein. In recent decades, however, it has become evident that a large fraction of the proteome, particularly in eukaryotes, stays disordered in solution and these proteins are termed as intrinsically disordered proteins (IDPs). Also, a vast majority of human proteomes have been reported to contain substantially long disordered regions, called intrinsically disordered regions (IDRs), in addition to the structurally ordered regions. IDPs exist in an ensemble of conformations and the conformational flexibility enables IDPs to achieve functional diversity. IDPs (and IDRs) are found to be important players in cell signaling, where biological membranes act as anchors for signaling cascades. Therefore, IDPs modulate the membrane architectures, at the same time membrane composition also affects the binding of IDPs. Because of intrinsic disorders, misfolding of IDPs often leads to formation of oligomers, protofibrils and mature fibrils through progressive self-association. Accumulation of amyloid-like aggregates of some of the IDPs is a known causative agent for numerous diseases. In this chapter we highlight recent advances in understanding membrane interactions of some of the intrinsically disordered proteins involved in the pathogenesis of human diseases.

Backbone Dynamics of the TAZ1 Domain of the Creb-Binding Protein Modulate Competition Between Disordered Ligands

Intrinsically disordered proteins (IDPs) play important roles in a multitude of cellular processes; however, the mechanisms by which disordered proteins carry out their diverse roles as modulators of cellular signaling are not yet completely understood. IDPs utilize a wide range of mechanisms to interact with folded proteins within the cell, and in many cases, must compete for binding of protein interaction partners. Yet, little is known about the contributions of the folded proteins to the binding and specificity of interactions with multiple disordered ligands. Are folded protein interaction partners simply scaffolds for IDP binding or do they undergo structural and dynamic changes to accommodate different disordered ligands? Using the folded TAZ1 domain of the transcriptional coactivator CBP and the disordered proteins HIF-1α and CITED2 as an example, we demonstrate that conformational dynamics and structural changes of TAZ1 are critical for binding and exchange of different IDPs. CITED2 competes with HIF-1α for TAZ1 binding through an extremely efficient unidirectional allosteric switch that relies not only on the physicochemical properties of the disordered proteins but also on the structure and dynamics of TAZ1. To gain further mechanistic insight into this process, we used NMR relaxation methods to obtain a detailed description of the backbone dynamics that contribute to TAZ1 binding and competition. We found that TAZ1 displays altered backbone dynamics in its free and bound states, with changes in TAZ1 dynamics observed not only in the HIF-1α and CITED2 binding sites but also in regions that differ in structure when bound to HIF-1α or CITED2. These data suggest that backbone dynamics in TAZ1 play a role in modulating binding of disordered ligands and highlight the importance of characterizing both folded and disordered partners in molecular interactions.

Measuring and Analyzing Binding Kinetics of Coupled Folding and Binding Reactions Under Pseudo-First-Order Conditions.

Many intrinsically disordered proteins (IDPs) adopt a well-defined structure upon binding to their interaction partners. Kinetic characterization is a requirement for the investigation of the dynamics and mechanisms of these folding-upon-binding reactions. Here a protocol is described for the investigation of binding kinetics of bimolecular binding and folding reactions of IDPs to their ligand partner under pseudo-first-order conditions using stopped-flow mixing and fluorescence detection.

Dissecting the Energetics of Intrinsically Disordered Proteins via a Hybrid Experimental and Computational Approach.

Intrinsically disordered proteins (IDPs) play important roles in biology, but little is known about the energetics of their inter-residue interactions. Methods that have been successfully applied to analyze the energetics of globular proteins are not applicable to the fluctuating partially ordered ensembles populated by IDPs. A combined computational experimental strategy is introduced for analyzing the energetic role of individual residues in the free state of IDPs. The approach combines experimental measurements of the binding of wild-type and mutant IDPs to their partners with alchemical free energy calculations of the structured complexes. These data allow quantitative information to be deduced about the free state via a thermodynamic cycle. The approach is validated by the analysis of the effects of mutations upon the binding free energy of the ovomucoid inhibitor third binding domain to its partners and is applied to the C-terminal domain of the measles virus nucleoprotein, a 125-residue IDP involved in the RNA transcription and replication of measles virus. The analysis reveals significant inter-residue interactions in the unbound IDP and suggests a biological role for them. The work demonstrates that advances in force fields and computational hardware have now led to the point where it is possible to develop methods, which integrate experimental and computational techniques to reveal insights that cannot be studied using either technique alone.

Three Color Single-Molecule FRET of Fast IDP Binding and Folding

Molecular interactions of intrinsically disordered proteins (IDPs) are different from those between folded molecules because IDP binding involves large convormational changes. To understand the mechanism of IDP binding, three-color Förster resonance energy transfer (FRET) will be very useful because both the interaction between two molecules and conformational changes can be probed simultaneously. In this work, we investigated binding of a disordered protein, the transactivation domain (TAD) of p53 and one of its binding partners, nuclear coactivator binding domain (NCBD) of CBP. TAD labeled with Alexa488 (donor) and Alexa594 (acceptor 1, A1) site-specifically was immobilized on a glass surface and incubated with CF680R (acceptor 2, A2)-labeled NCBD in solution. To determine all three FRET efficiencies, alternating excitation of the donor and A1 is typically used. However, in binding experiments, direct excitation of A2 attached to NCBD in solution by A1 excitation laser produces a high background signal. Therefore, we devised a method using a single donor excitation that determines all three FRET efficiencies by utilizing incomplete labeling of A2. When TAD binds to unlabled NCBD, the FRET efficiency between the donor and A1 (E1) can be determined. Using this information, it is possible to determine the other two FRET efficiencies in principle. However, due to the fast binding and dissociation kinetics, the bound and unbound states are not well separated in binned FRET efficiency trajectories (1 ms bin time), which prevents pre-determination of E1. We employed the maximum likelihood method developed by Gopich and Szabo that analyzes photon trajectories directly without binning the data. By globally analyzing the data using a kinetic model that includes binding to both labeled and unlabled NCBD, rate coefficients of the sub-millisecond kinetics and all three FRET efficiencies were determined accurately.

Diffusion-limited association of disordered protein by non-native electrostatic interactions

Intrinsically disordered proteins (IDPs) usually fold during binding to target proteins. In contrast to interactions between folded proteins, this additional folding step makes the binding process more complex. Understanding the mechanism of coupled binding and folding of IDPs requires analysis of binding pathways that involve formation of the transient complex (TC). However, experimental characterization of TC is challenging because it only appears for a very brief period during binding. Here, we use single-molecule fluorescence spectroscopy to investigate the mechanism of diffusion-limited association of an IDP. A large enhancement of the association rate is observed due to the stabilization of TC by non-native electrostatic interactions. Moreover, photon-by-photon analysis reveals that the lifetime of TC for IDP binding is at least two orders of magnitude longer than that for binding of two folded proteins. This result suggests the long lifetime of TC is generally required for folding of IDPs during binding processes.

Dynamics, Conformational Entropy, and Frustration in Protein-Protein Interactions Involving an Intrinsically Disordered Protein Domain.

Intrinsically disordered proteins (IDPs) are abundant in the eukaryotic proteome. However, little is known about the role of subnanosecond dynamics and the conformational entropy that it represents in protein-protein interactions involving IDPs. Using nuclear magnetic resonance side chain and backbone relaxation, stopped-flow kinetics, isothermal titration calorimetry, and computational studies, we have characterized the interaction between the globular TAZ1 domain of the CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2). We show that the TAZ1/TAD-STAT2 complex retains considerable subnanosecond motions, with TAD-STAT2 undergoing only a partial disorder-to-order transition. We report here the first experimental determination of the conformational entropy change for both binding partners in an IDP binding interaction and find that the total change even exceeds in magnitude the binding enthalpy and is comparable to the contribution from the hydrophobic effect, demonstrating its importance in the binding energetics. Furthermore, we show that the conformational entropy change for TAZ1 is also instrumental in maintaining a biologically meaningful binding affinity. Strikingly, a spatial clustering of very high amplitude motions and a cluster of more rigid sites in the complex exist, which through computational studies we found to overlap with regions that experience energetic frustration and are less frustrated, respectively. Thus, the residual dynamics in the bound state could be necessary for faster dissociation, which is important for proteins that interact with multiple binding partners.

Looking at the Disordered Proteins through the Computational Microscope.

Intrinsically disordered proteins (IDPs) have attracted wide interest over the past decade due to their surprising prevalence in the proteome and versatile roles in cell physiology and pathology. A large selection of IDPs has been identified as potential targets for therapeutic intervention. Characterizing the structure–function relationship of disordered proteins is therefore an essential but daunting task, as these proteins can adapt transient structure, necessitating a new paradigm for connecting structural disorder to function. Molecular simulation has emerged as a natural complement to experiments for atomic-level characterizations and mechanistic investigations of this intriguing class of proteins. The diverse range of length and time scales involved in IDP function requires performing simulations at multiple levels of resolution. In this Outlook, we focus on summarizing available simulation methods, along with a few interesting example applications. We also provide an outlook on how these simulation methods can be further improved in order to provide a more accurate description of IDP structure, binding, and assembly.

Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy.

Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions, or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state. Knowledge of the dynamics of IDP complexes is of fundamental importance to understand how IDPs engage in highly specific interactions without concomitantly high binding affinity. Here, we combine rotating-frame R1ρ, Carr-Purcell-Meiboom Gill relaxation dispersion as well as chemical exchange saturation transfer to decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interaction modes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.

Exploring the Role of Flexibility in Binding Kinetics and Affinity of pKID-Kix through Coarse Grained Simulations

Physical properties that distinguish Intrinsically Disordered Proteins (IDPs) from structured proteins include increased association rates and the formation of bound complexes with high specificity and low affinity. In this work, we investigate the influence of flexibility on binding kinetics and thermodynamics of IDPs through coarse grained simulations. Using pKID-KIX complex as an example, we illustrate how coupled folding and binding allows the IDP to have both fast binding and unbinding rates together with low binding affinity. In particular, we show that while flexibility moderately increases binding rate (consistent with the fly-casting mechanism), the unbinding rate is enhanced to a greater extent, resulting in a low affinity complex. Analysis of equilibrium trajectories show that a flexible protein forms contacts incrementally throughout binding and that orientational constraints are satisfied gradually; both of which encourage a lower free energy barrier controlling binding and unbinding kinetics. Our study highlights the importance that flexibility has not only on an IDP's binding rate, but also on its ability to form low affinity complexes with sufficiently fast unbinding kinetics essential to their function.

Increasing the Chemical-Shift Dispersion of Unstructured Proteins with a Covalent Lanthanide Shift Reagent.

The study of intrinsically disordered proteins (IDPs) by NMR often suffers from highly overlapped resonances that prevent unambiguous chemical‐shift assignments, and data analysis that relies on well‐separated resonances. We present a covalent paramagnetic lanthanide‐binding tag (LBT) for increasing the chemical‐shift dispersion and facilitating the chemical‐shift assignment of challenging, repeat‐containing IDPs. Linkage of the DOTA‐based LBT to a cysteine residue induces pseudo‐contact shifts (PCS) for resonances more than 20 residues from the spin‐labeling site. This leads to increased chemical‐shift dispersion and decreased signal overlap, thereby greatly facilitating chemical‐shift assignment. This approach is applicable to IDPs of varying sizes and complexity, and is particularly helpful for repeat‐containing IDPs and low‐complexity regions. This results in improved efficiency for IDP analysis and binding studies.

Binding of Disordered Peptides to Kelch: Insights from Enhanced Sampling Simulations.

Keap1 protein plays an essential role in regulating cellular oxidative stress response and is a crucial binding hub for multiple proteins, several of which are intrinsically disordered proteins (IDP). Among Kelch's IDP binding partners, NRF2 and PTMA are the two most interesting cases. They share a highly similar binding motif; however, NRF2 binds to Kelch with a binding affinity of approximately 100-fold higher than that of PTMA. In this study, we perform an exhaustive sampling composed of 6 μs well-tempered metadynamics and 2 μs unbiased molecular dynamics (MD) simulations aiming at characterizing the binding mechanisms and structural properties of these two peptides. Our results agree with previous experimental observations that PTMA is remarkably more disordered than NRF2 in both the free and bound states. This explains PTMA's lower binding affinity. Our extensive sampling also provides valuable insights into the vast conformational ensembles of both NRF2 and PTMA, supports the hypothesis of coupled folding-binding, and confirms the essential role of linear motifs in IDP binding.

Effect of O-Linked Glycosylation on the Equilibrium Structural Ensemble of Intrinsically Disordered Polypeptides.

Glycosylation is one of the most common post-translational modifications (PTMs), which provides a large proteome diversity. Previous work on glycosylation of globular proteins has revealed remarkable effects of glycosylation on protein function, altering the folding stability and structure and/or altering the protein surface which affects their binding characteristics. Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) of large proteins are also frequently glycosylated, yet how glycosylation affects their function remains to be elucidated. An important open question is, does glycosylation affect IDP structure or binding characteristics or both? In this work, we particularly address the structural effects of O-linked glycosylation by investigating glycosylated and unglycosylated forms of two different IDPs, tau174-183 and human islet amyloid polypeptide (hIAPP), by all-atom explicit solvent simulations. We simulate these IDPs in aqueous solution for O-linked glycosylated and unglycosylated forms by employing two modern all-atom force fields for which glycan parameters are also available. We find that O-linked glycosylation only has a modest effect on equilibrium structural ensembles of IDPs, for the cases studied here, which suggests that the functional role of glycosylation may be primarily exerted by modulation of the protein binding characteristics rather than structure.

Dihedral angle entropy measures for intrinsically disordered proteins.

Protein stability is based on a delicate balance between energetic and entropic factors. Intrinsically disordered proteins (IDPs) interacting with a folded partner protein in the act of binding can order the IDP to form the correct functional interface by decrease in the overall free energy. In this work, we evaluate the part of the entropic cost of ordering an IDP arising from their dihedral states. The IDP studied is a leucine zipper dimer that we simulate with molecular dynamics and find that it does show disorder in six phi and psi dihedral angles of the N terminal sequence of one monomer. Essential to ascertain is the degree of disorder in the IDP, and we do so by considering the entire, discretized probability distribution function of N dihedrals with M conformers per dihedral. A compositional clustering method is introduced, whereby the NS = N(M) states are formed from the Cartesian product of each dihedral's conformational space. Clustering is carried out with a version of a k-means algorithm that accounts for the circular nature of dihedral angles. For the 12 dihedrals each found to have three conformers, among the resulting 531441 states, their populations show that the first 100 (500) most populated states account for ∼65% (∼90%) of the entire population, indicating that there are strong dependencies among the dihedrals' conformations. These state populations are used to evaluate a Kullback-Leibler divergence entropy measure and obtain the dihedral configurational entropy S. At 300 K, TS ∼ 3 kcal/mol, showing that IDP entropy, while roughly half that would be expected from independently distributed dihedrals, can be a decisive contributor to the free energy of this IDP binding and ordering.

Quantitative biophysical characterization of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are broadly defined as protein regions that do not cooperatively fold into a spatially or temporally stable structure. Recent research strongly supports the hypothesis that a conserved functional role for structural disorder renders IDPs uniquely capable of functioning in biological processes such as cellular signaling and transcription. Recently, the frequency of application of rigorous mechanistic biochemistry and quantitative biophysics to disordered systems has increased dramatically. For example, the launch of the Protein Ensemble Database (pE-DB) demonstrates that the potential now exists to refine models for the native state structure of IDPs using experimental data. However, rigorous assessment of which observables place the strongest and least biased constraints on those ensembles is now needed. Most importantly, the past few years have seen strong growth in the number of biochemical and biophysical studies attempting to connect structural disorder with function. From the perspective of equilibrium thermodynamics, there is a clear need to assess the relative significance of hydrophobic versus electrostatic forces in IDP interactions, if it is possible to generalize at all. Finally, kinetic mechanisms that invoke conformational selection and/or induced fit are often used to characterize coupled IDP folding and binding, although application of these models is typically built upon thermodynamic observations. Recently, the reaction rates and kinetic mechanisms of more intrinsically disordered systems have been tested through rigorous kinetic experiments. Motivated by these exciting advances, here we provide a review and prospectus for the quantitative study of IDP structure, thermodynamics, and kinetics.