Enthalpy–Entropy Compensation in the Promiscuous Interaction of an Intrinsically Disordered Protein with Homologous Protein Partners

Intrinsically disordered proteins (IDPs) can engage in promiscuous interactions with their protein targets; however, it is not clear how this feature is encoded in the primary sequence of the IDPs and to what extent the surface properties and the shape of the binding cavity dictate the binding mode and the final bound conformation. Here we show, using a combination of nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), that the promiscuous interaction of the intrinsically disordered regulatory domain of the mitogen-activated protein kinase kinase MKK4 with p38α and JNK1 is facilitated by folding-upon-binding into two different conformations, despite the high sequence conservation and structural homology between p38α and JNK1. Our results support a model whereby the specific surface properties of JNK1 and p38α dictate the bound conformation of MKK4 and that enthalpy–entropy compensation plays a major role in maintaining comparable binding affinities for MKK4 towards the two kinases.

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Generation of the configurational ensemble of an intrinsically disordered protein from unbiased molecular dynamics simulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1907251116 ◽

2019 ◽

Vol 116 (41) ◽

pp. 20446-20452 ◽

Cited By ~ 9

Author(s):

Utsab R. Shrestha ◽

Puneet Juneja ◽

Qiu Zhang ◽

Viswanathan Gurumoorthy ◽

Jose M. Borreguero ◽

...

Keyword(s):

Molecular Dynamics ◽

Intrinsically Disordered Proteins ◽

Chemical Shifts ◽

Intrinsically Disordered Protein ◽

Physical Models ◽

Dynamics Simulation ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Eukaryotic Proteomes ◽

Heterogeneous Ensemble

Intrinsically disordered proteins (IDPs) are abundant in eukaryotic proteomes, play a major role in cell signaling, and are associated with human diseases. To understand IDP function it is critical to determine their configurational ensemble, i.e., the collection of 3-dimensional structures they adopt, and this remains an immense challenge in structural biology. Attempts to determine this ensemble computationally have been hitherto hampered by the necessity of reweighting molecular dynamics (MD) results or biasing simulation in order to match ensemble-averaged experimental observables, operations that reduce the precision of the generated model because different structural ensembles may yield the same experimental observable. Here, by employing enhanced sampling MD we reproduce the experimental small-angle neutron and X-ray scattering profiles and the NMR chemical shifts of the disordered N terminal (SH4UD) of c-Src kinase without reweighting or constraining the simulations. The unbiased simulation results reveal a weakly funneled and rugged free energy landscape of SH4UD, which gives rise to a heterogeneous ensemble of structures that cannot be described by simple polymer theory. SH4UD adopts transient helices, which are found away from known phosphorylation sites and could play a key role in the stabilization of structural regions necessary for phosphorylation. Our findings indicate that adequately sampled molecular simulations can be performed to provide accurate physical models of flexible biosystems, thus rationalizing their biological function.

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Enhancing Binding Affinity of an Intrinsically Disordered Protein by α-Methylation of Key Amino Acid Residues

10.26434/chemrxiv.10113128 ◽

2019 ◽

Author(s):

Valentin Bauer ◽

Boris Schmidtgall ◽

Gergő Gógl ◽

Jozica Dolenc ◽

Judit Osz ◽

...

Keyword(s):

Amino Acids ◽

Protein Interactions ◽

Intrinsically Disordered Proteins ◽

Intrinsically Disordered Protein ◽

Selective Inhibition ◽

Disordered Proteins ◽

Creb Binding Protein ◽

Alpha Helices ◽

Intrinsically Disordered ◽

Order Of Magnitude

Intrinsically disordered proteins (IDPs), which undergo folding upon binding to their targets, are critical players in protein interaction networks. Here we demonstrate that incorporation of non-canonical alpha-methylated amino acids into the unstructured activation domain of the transcriptional coactivator ACTR can stabilize helical conformations and strengthen binding interactions with the nuclear coactivator binding domain (NCBD) of CREB-binding protein (CBP). A combinatorial alpha-methylation scan of the ACTR sequence converged on two substitutions at positions 1055 and 1076 that increase affinity for both NCBD and the full length 270 kDa CBP by one order of magnitude. The first X-ray structure of the modified ACTR domain bound to NCBD revealed that the key alpha-methylated amino acids were localized within alpha-helices. Biophysical studies showed that the observed changes in binding energy are the result of long-range interactions and redistribution of enthalpy and entropy. This proof-of-concept study establishes a potential strategy for selective inhibition of protein-protein interactions involving IDPs in cells.<br>

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An intrinsically disordered protein, CP12: jack of all trades and master of the Calvin cycle

Biochemical Society Transactions ◽

10.1042/bst20120097 ◽

2012 ◽

Vol 40 (5) ◽

pp. 995-999 ◽

Cited By ~ 41

Author(s):

Brigitte Gontero ◽

Stephen C. Maberly

Keyword(s):

Amino Acids ◽

Stress Responses ◽

Intrinsically Disordered Proteins ◽

Calvin Cycle ◽

Intrinsically Disordered Protein ◽

Disulfide Bridge ◽

Co2 Assimilation ◽

Disordered Proteins ◽

Dimensional Structure ◽

Intrinsically Disordered

Many proteins contain disordered regions under physiological conditions and lack specific three-dimensional structure. These are referred to as IDPs (intrinsically disordered proteins). CP12 is a chloroplast protein of approximately 80 amino acids and has a molecular mass of approximately 8.2–8.5 kDa. It is enriched in charged amino acids and has a small number of hydrophobic residues. It has a high proportion of disorder-promoting residues, but has at least two (often four) cysteine residues forming one (or two) disulfide bridge(s) under oxidizing conditions that confers some order. However, CP12 behaves like an IDP. It appears to be universally distributed in oxygenic photosynthetic organisms and has recently been detected in a cyanophage. The best studied role of CP12 is its regulation of the Calvin cycle responsible for CO2 assimilation. Oxidized CP12 forms a supramolecular complex with two key Calvin cycle enzymes, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and PRK (phosphoribulokinase), down-regulating their activity. Association–dissociation of this complex, induced by the redox state of CP12, allows the Calvin cycle to be inactive in the dark and active in the light. CP12 is promiscuous and interacts with other enzymes such as aldolase and malate dehydrogenase. It also plays other roles in plant metabolism such as protecting GAPDH from inactivation and scavenging metal ions such as copper and nickel, and it is also linked to stress responses. Thus CP12 seems to be involved in many functions in photosynthetic cells and behaves like a jack of all trades as well as being a master of the Calvin cycle.

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Supramolecular Fuzziness of Intracellular Liquid Droplets: Liquid–Liquid Phase Transitions, Membrane-Less Organelles, and Intrinsic Disorder

Molecules ◽

10.3390/molecules24183265 ◽

2019 ◽

Vol 24 (18) ◽

pp. 3265 ◽

Cited By ~ 12

Author(s):

Vladimir N. Uversky

Keyword(s):

Phase Transitions ◽

Liquid Phase ◽

Intrinsically Disordered Proteins ◽

Intrinsically Disordered Protein ◽

Disordered Proteins ◽

Bacterial Cells ◽

Liquid Droplets ◽

Intrinsically Disordered ◽

Wide Range ◽

Characteristic Features

Cells are inhomogeneously crowded, possessing a wide range of intracellular liquid droplets abundantly present in the cytoplasm of eukaryotic and bacterial cells, in the mitochondrial matrix and nucleoplasm of eukaryotes, and in the chloroplast’s stroma of plant cells. These proteinaceous membrane-less organelles (PMLOs) not only represent a natural method of intracellular compartmentalization, which is crucial for successful execution of various biological functions, but also serve as important means for the processing of local information and rapid response to the fluctuations in environmental conditions. Since PMLOs, being complex macromolecular assemblages, possess many characteristic features of liquids, they represent highly dynamic (or fuzzy) protein–protein and/or protein–nucleic acid complexes. The biogenesis of PMLOs is controlled by specific intrinsically disordered proteins (IDPs) and hybrid proteins with ordered domains and intrinsically disordered protein regions (IDPRs), which, due to their highly dynamic structures and ability to facilitate multivalent interactions, serve as indispensable drivers of the biological liquid–liquid phase transitions (LLPTs) giving rise to PMLOs. In this article, the importance of the disorder-based supramolecular fuzziness for LLPTs and PMLO biogenesis is discussed.

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NSP 11 of SARS-CoV-2 is an Intrinsically Disordered Protein

10.1101/2020.10.07.330068 ◽

2020 ◽

Cited By ~ 1

Author(s):

Kundlik Gadhave ◽

Prateek Kumar ◽

Ankur Kumar ◽

Taniya Bhardwaj ◽

Neha Garg ◽

...

Keyword(s):

Amino Acid ◽

Intrinsically Disordered Proteins ◽

Conformational Dynamics ◽

Alpha Helix ◽

Intrinsically Disordered Protein ◽

Disordered Proteins ◽

Helical Conformation ◽

Structural Protein ◽

Intrinsically Disordered ◽

Protein Size

AbstractThe intrinsically disordered proteins/regions (IDPs/IDPRs) are known to be responsible for multiple cellular processes and are associated with many chronic diseases. In viruses, the existence of disordered proteome is also proven and are related with its conformational dynamics inside the host. The SARS-CoV-2 virus has a large proteome, in which, structure and functions of many proteins are not known as of yet. Previously, we have investigated the dark proteome of SARS-CoV-2. However, the disorder status of non-structural protein 11 (nsp11) was not possible because of very small in size, just 13 amino acid long, and for most of the IDP predictors, the protein size should be at least 30 amino acid long. Also, the structural dynamics and function status of nsp11 was not known. Hence, we have performed extensive experimentation on nsp11. Our results, based on the Circular dichroism spectroscopy gives characteristic disordered spectrum for IDPs. Further, we investigated the conformational behaviour of nsp11 in the presence of membrane mimetic environment, alpha helix inducer, and natural osmolyte. In the presence of negatively charged and neutral liposomes, nsp11 remains disordered. However, with SDS micelle, it adopted an α-helical conformation, suggesting the helical propensity of nsp11. At the end, we again confirmed the IDP behaviour of nsp11 using molecular dynamics simulations.

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Aptamers Selected for Recognizing Amyloid β-protein—A Case for Cautious Optimism

10.20944/preprints201802.0047.v1 ◽

2018 ◽

Author(s):

Farid Rahimi

Keyword(s):

Intrinsically Disordered Proteins ◽

Scientific Evidence ◽

Protein A ◽

Intrinsically Disordered Protein ◽

Disordered Proteins ◽

Full Potential ◽

Amyloid Beta Protein ◽

Intrinsically Disordered ◽

Metastable Structures

Aptamers are versatile oligonucleotide ligands used for molecular recognition of diverse targets. However, application of aptamers to the field of amyloid β-protein (Aβ) has been limited so far. Aβ is an intrinsically disordered protein that exists in a dynamic conformational equilibrium, presenting time-dependent ensembles of short-lived, metastable structures and assemblies that have been generally difficult to isolate and characterize. Moreover, despite understanding of potential physiological roles of Aβ, this peptide has been linked to the pathogenesis of Alzheimer disease, and its pathogenic roles remain controversial. Accumulated scientific evidence thus far highlights undesirable or nonspecific interactions between selected aptamers and different Aβ assemblies likely due to metastable nature of Aβ or inherent affinity of RNA oligonucleotides to β-sheet-rich fibrillar structures of amyloidogenic proteins. Accordingly, lessons drawn from Aβ–aptamer studies emphasize that purity and uniformity of the protein target and rigorous characterization of aptamers’ specificity are important for realizing and garnering the full potential of aptamers selected for recongizing Aβ or other intrinsically disordered proteins. This review summarizes studies of aptamers selected for recognizing different Aβ assemblies and highlights controversies, difficulties, and limitations of such studies.

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Exploring Energy Landscapes of Intrinsically Disordered Proteins: Insights into Functional Mechanisms

10.1101/2021.01.28.428595 ◽

2021 ◽

Author(s):

Antonio B. Oliveira ◽

Xingcheng Lin ◽

Prakash Kulkarni ◽

José N. Onuchic ◽

Susmita Roy ◽

...

Keyword(s):

Intrinsically Disordered Proteins ◽

Energy Landscape ◽

3D Structure ◽

Intrinsically Disordered Protein ◽

Terminal Segment ◽

Disordered Proteins ◽

Conformational Ensemble ◽

Energy Landscapes ◽

Reaction Coordinates ◽

Intrinsically Disordered

AbstractIntrinsically disordered proteins (IDPs) lack a rigid 3D structure and populate a polymorphic ensemble of conformations. Because of the lack of a reference conformation, their energy landscape representation in terms of reaction coordinates presents a daunting challenge. Here, our newly developed Energy Landscape Visualization Method (ELViM), a reaction coordinate-free approach, shows its prime application to explore frustrated energy landscapes of an intrinsically disordered protein, Prostate-Associated Gene 4 (PAGE4). PAGE4 is a transcriptional coactivator that potentiates the oncogene c-Jun. Two kinases, namely HIPK1 and CLK2, phosphorylate PAGE4 generating variants phosphorylated at different serine/threonine residues (HIPK1-PAGE4 and CLK2-PAGE4, respectively) with opposing functions. While HIPK1-PAGE4 predominantly phosphorylates Thr51 and potentiates c-Jun, CLK2-PAGE4 hyper-phosphorylates PAGE4 and attenuates transactivation. To understand the underlying mechanisms of conformational diversity among different phosphoforms, we have analyzed their atomistic trajectories simulated using AWSEM forcefield and the energy landscapes were elucidated using ELViM. This method allows us to identify and compare the population distributions of different conformational ensembles of PAGE4 phosphoforms using the same effective phase space. The results reveal a predominant conformational ensemble with an extended C-terminal segment of WT PAGE4, which exposes a functional residue Thr51, implying its potential of undertaking a fly-casting mechanism while binding to its cognate partner. In contrast, for HIPK1-PAGE4, a compact conformational ensemble enhances its population sequestering phosphorylated-Thr51. This clearly explains the experimentally observed weaker affinity of HIPK1-PAGE4 for c-Jun. ELViM appears as a powerful tool especially to analyze the highly-frustrated energy landscape representation of IDPs where appropriate reaction coordinates are hard to apprehend.

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Design of Inhibitors of the Intrinsically Disordered Protein NUPR1: Balance between Drug Affinity and Target Function

Biomolecules ◽

10.3390/biom11101453 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1453

Author(s):

Bruno Rizzuti ◽

Wenjun Lan ◽

Patricia Santofimia-Castaño ◽

Zhengwei Zhou ◽

Adrián Velázquez-Campoy ◽

...

Keyword(s):

Intrinsically Disordered Proteins ◽

Nuclear Translocation ◽

Alkyl Chain ◽

Biological Effects ◽

Target Function ◽

Disordered Proteins ◽

Ductal Adenocarcinoma ◽

Titration Calorimetry ◽

Intrinsically Disordered

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their physiological ubiquity and their prevalence in various diseases, including cancer. NUPR1 is an IDP that localizes throughout the whole cell, and is involved in the development and progression of several tumors. We have previously repurposed trifluoperazine (TFP) as a drug targeting NUPR1 and, by using a ligand-based approach, designed the drug ZZW-115 starting from the TFP scaffold. Such derivative compound hinders the development of pancreatic ductal adenocarcinoma (PDAC) in mice, by hampering nuclear translocation of NUPR1. Aiming to further improve the activity of ZZW-115, here we have used an indirect drug design approach to modify its chemical features, by changing the substituent attached to the piperazine ring. As a result, we have synthesized a series of compounds based on the same chemical scaffold. Isothermal titration calorimetry (ITC) showed that, with the exception of the compound preserving the same chemical moiety at the end of the alkyl chain as ZZW-115, an increase of the length by a single methylene group (i.e., ethyl to propyl) significantly decreased the affinity towards NUPR1 measured in vitro, whereas maintaining the same length of the alkyl chain and adding heterocycles favored the binding affinity. However, small improvements of the compound affinity towards NUPR1, as measured by ITC, did not result in a corresponding improvement in their inhibitory properties and in cellulo functions, as proved by measuring three different biological effects: hindrance of the nuclear translocation of the protein, sensitization of cells against DNA damage mediated by NUPR1, and prevention of cancer cell growth. Our findings suggest that a delicate compromise between favoring ligand affinity and controlling protein function may be required to successfully design drugs against NUPR1, and likely other IDPs.

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New technologies to analyse protein function: an intrinsic disorder perspective

F1000Research ◽

10.12688/f1000research.20867.1 ◽

2020 ◽

Vol 9 ◽

pp. 101 ◽

Cited By ~ 2

Author(s):

Vladimir N. Uversky

Keyword(s):

Protein Function ◽

Intrinsically Disordered Proteins ◽

New Technologies ◽

Intrinsically Disordered Protein ◽

Intrinsic Disorder ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Disordered Protein ◽

Protein Functions ◽

Require Structure

Functions of intrinsically disordered proteins do not require structure. Such structure-independent functionality has melted away the classic rigid “lock and key” representation of structure–function relationships in proteins, opening a new page in protein science, where molten keys operate on melted locks and where conformational flexibility and intrinsic disorder, structural plasticity and extreme malleability, multifunctionality and binding promiscuity represent a new-fangled reality. Analysis and understanding of this new reality require novel tools, and some of the techniques elaborated for the examination of intrinsically disordered protein functions are outlined in this review.

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Affinity versus specificity in coupled binding and folding reactions

Protein Engineering Design and Selection ◽

10.1093/protein/gzz020 ◽

2019 ◽

Author(s):

Stefano Gianni ◽

Per Jemth

Keyword(s):

Protein Interactions ◽

Intrinsically Disordered Proteins ◽

Intrinsically Disordered Protein ◽

High Specificity ◽

Disordered Proteins ◽

Protein Protein Interactions ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Interaction Interface ◽

Disordered Regions

Abstract Intrinsically disordered protein regions may fold upon binding to an interaction partner. It is often argued that such coupled binding and folding enables the combination of high specificity with low affinity. The basic tenet is that an unfavorable folding equilibrium will make the overall binding weaker while maintaining the interaction interface. While theoretically solid, we argue that this concept may be misleading for intrinsically disordered proteins. In fact, experimental evidence suggests that interactions of disordered regions usually involve extended conformations. In such cases, the disordered region is exceptionally unlikely to fold into a bound conformation in the absence of its binding partner. Instead, these disordered regions can bind to their partners in multiple different conformations and then fold into the native bound complex, thus, if anything, increasing the affinity through folding. We concede that (de)stabilization of native structural elements such as helices will modulate affinity, but this could work both ways, decreasing or increasing the stability of the complex. Moreover, experimental data show that intrinsically disordered binding regions display a range of affinities and specificities dictated by the particular side chains and length of the disordered region and not necessarily by the fact that they are disordered. We find it more likely that intrinsically disordered regions are common in protein–protein interactions because they increase the repertoire of binding partners, providing an accessible route to evolve interactions rather than providing a stability–affinity trade-off.

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