Multisite Phosphorylation and Binding Alter Conformational Dynamics of the 4E-BP2 Protein

Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions, but detailed structural/dynamics characterization of their ensembles remain challenging, both in isolation and they form dynamic fuzzy complexes. Such is the case for mRNA cap-dependent translation initiation, which is regulated by the interaction of the predominantly folded eukaryotic initiation factor 4E (eIF4E) with the intrinsically disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Single-molecule Forster resonance energy transfer showed that the conformational changes of 4E-BP2 induced by binding to eIF4E are non-uniform along the sequence; while a central region containing both motifs that bind to eIF4E expands and becomes stiffer, the C-terminal region is less affected. Fluorescence anisotropy decay revealed a nonuniform segmental flexibility around six different labelling sites along the chain. Dynamic quenching of these fluorescent probes by intrinsic aromatic residues measured via fluorescence correlation spectroscopy report on transient intra- and inter-molecular contacts on nanosecond-microsecond timescales. Upon hyperphosphorylation, which induces folding of ~40 residues in 4E-BP2, the quenching rates decreased at labelling sites closest to the phosphorylation sites and within the folded domain, and increased at the other sites. The chain dynamics around sites in the C-terminal region far away from the two binding motifs were significantly reduced upon binding to eIF4E, suggesting that this region is also involved in the highly dynamic 4E-BP2:eIF4E complex. Our time-resolved fluorescence data paint a sequence-level rigidity map of three states of 4E-BP2 differing in phosphorylation or binding status and distinguish regions that form contacts with eIF4E. This study adds complementary structural and dynamics information to recent studies of 4E-BP2, and it constitutes an important step towards a mechanistic understanding of this important IDP via integrative modelling.

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Current Challenges and Limitations in the Studies of Intrinsically Disordered Proteins in Neurodegenerative Diseases by Computer Simulations

Current Alzheimer Research ◽

10.2174/1567205017666201109094908 ◽

2020 ◽

Vol 17 ◽

Author(s):

Ibrahim Yagiz Akbayrak ◽

Sule Irem Caglayan ◽

Zilan Ozcan ◽

Vladimir N. Uversky ◽

Orkid Coskuner-Weber

Keyword(s):

Neurodegenerative Diseases ◽

Computer Simulations ◽

Conformational Changes ◽

Intrinsically Disordered Proteins ◽

Disordered Proteins ◽

Computational Studies ◽

Accurate Data ◽

Aggregation Propensity ◽

Intrinsically Disordered ◽

Future Developments

: Experiments face challenges in the analysis of intrinsically disordered proteins in solution due to fast conformational changes and enhanced aggregation propensity. Computational studies complement experiments, being widely used in the analyses of intrinsically disordered proteins, especially those positioned at the centers of neurodegenerative diseases. However, recent investigations – including our own – revealed that computer simulations face significant challenges and limitations themselves. In this review, we introduced and discussed some of the scientific challenges and limitations of computational studies conducted on intrinsically disordered proteins. We also outlined the importance of future developments in the areas of computational chemistry and computational physics that would be needed for generating more accurate data for intrinsically disordered proteins from computer simulations. Additional theoretical strategies that can be developed are discussed herein.

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From folding to function: complex macromolecular reactions unraveled one-by-one with optical tweezers

Essays in Biochemistry ◽

10.1042/ebc20200024 ◽

2021 ◽

Author(s):

Pétur O. Heidarsson ◽

Ciro Cecconi

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Structural Changes ◽

Intrinsically Disordered Proteins ◽

Conformational Dynamics ◽

Disordered Proteins ◽

Kinase Activation ◽

Intrinsically Disordered ◽

Complex Protein ◽

Methodological Principles

Abstract Single-molecule manipulation with optical tweezers has uncovered macromolecular behaviour hidden to other experimental techniques. Recent instrumental improvements have made it possible to expand the range of systems accessible to optical tweezers. Beyond focusing on the folding and structural changes of isolated single molecules, optical tweezers studies have evolved into unraveling the basic principles of complex molecular processes such as co-translational folding on the ribosome, kinase activation dynamics, ligand–receptor binding, chaperone-assisted protein folding, and even dynamics of intrinsically disordered proteins (IDPs). In this mini-review, we illustrate the methodological principles of optical tweezers before highlighting recent advances in studying complex protein conformational dynamics – from protein synthesis to physiological function – as well as emerging future issues that are beginning to be addressed with novel approaches.

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Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and folding

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1512799112 ◽

2015 ◽

Vol 112 (31) ◽

pp. 9614-9619 ◽

Cited By ~ 138

Author(s):

Munehito Arai ◽

Kenji Sugase ◽

H. Jane Dyson ◽

Peter E. Wright

Keyword(s):

Intrinsically Disordered Proteins ◽

Terminal Region ◽

Relaxation Dispersion ◽

Disordered Proteins ◽

Conformational Ensemble ◽

Induced Fit ◽

Conformational Selection ◽

Intrinsically Disordered ◽

Factor C ◽

Binding Mechanisms

Intrinsically disordered proteins (IDPs) frequently function in protein interaction networks that regulate crucial cellular signaling pathways. Many IDPs undergo transitions from disordered conformational ensembles to folded structures upon binding to their cellular targets. Several possible binding mechanisms for coupled folding and binding have been identified: folding of the IDP after association with the target (“induced fit”), or binding of a prefolded state in the conformational ensemble of the IDP to the target protein (“conformational selection”), or some combination of these two extremes. The interaction of the intrinsically disordered phosphorylated kinase-inducible domain (pKID) of the cAMP-response element binding (CREB) protein with the KIX domain of a general transcriptional coactivator CREB-binding protein (CBP) provides an example of the induced-fit mechanism. Here we show by NMR relaxation dispersion experiments that a different intrinsically disordered ligand, the transactivation domain of the transcription factor c-Myb, interacts with KIX at the same site as pKID but via a different binding mechanism that involves elements of conformational selection and induced fit. In contrast to pKID, the c-Myb activation domain has a strong propensity for spontaneous helix formation in its N-terminal region, which binds to KIX in a predominantly folded conformation. The C-terminal region of c-Myb exhibits a much smaller helical propensity and likely folds via an induced-fit process after binding to KIX. We propose that the intrinsic secondary structure propensities of pKID and c-Myb determine their binding mechanisms, consistent with their functions as inducible and constitutive transcriptional activators.

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Structural and Dynamical Order of a Disordered Protein: Molecular Insights into Conformational Switching of PAGE4 at the Systems Level

Biomolecules ◽

10.3390/biom9020077 ◽

2019 ◽

Vol 9 (2) ◽

pp. 77 ◽

Cited By ~ 6

Author(s):

Xingcheng Lin ◽

Prakash Kulkarni ◽

Federico Bocci ◽

Nicholas Schafer ◽

Susmita Roy ◽

...

Keyword(s):

Intrinsically Disordered Proteins ◽

Conformational Dynamics ◽

Structural Plasticity ◽

Disordered Proteins ◽

Dimensional Structure ◽

Related Disorder ◽

Structural Ordering ◽

Intrinsically Disordered ◽

Collective Motions ◽

Folded Proteins

Folded proteins show a high degree of structural order and undergo (fairly constrained) collective motions related to their functions. On the other hand, intrinsically disordered proteins (IDPs), while lacking a well-defined three-dimensional structure, do exhibit some structural and dynamical ordering, but are less constrained in their motions than folded proteins. The larger structural plasticity of IDPs emphasizes the importance of entropically driven motions. Many IDPs undergo function-related disorder-to-order transitions driven by their interaction with specific binding partners. As experimental techniques become more sensitive and become better integrated with computational simulations, we are beginning to see how the modest structural ordering and large amplitude collective motions of IDPs endow them with an ability to mediate multiple interactions with different partners in the cell. To illustrate these points, here, we use Prostate-associated gene 4 (PAGE4), an IDP implicated in prostate cancer (PCa) as an example. We first review our previous efforts using molecular dynamics simulations based on atomistic AWSEM to study the conformational dynamics of PAGE4 and how its motions change in its different physiologically relevant phosphorylated forms. Our simulations quantitatively reproduced experimental observations and revealed how structural and dynamical ordering are encoded in the sequence of PAGE4 and can be modulated by different extents of phosphorylation by the kinases HIPK1 and CLK2. This ordering is reflected in changing populations of certain secondary structural elements as well as in the regularity of its collective motions. These ordered features are directly correlated with the functional interactions of WT-PAGE4, HIPK1-PAGE4 and CLK2-PAGE4 with the AP-1 signaling axis. These interactions give rise to repeated transitions between (high HIPK1-PAGE4, low CLK2-PAGE4) and (low HIPK1-PAGE4, high CLK2-PAGE4) cell phenotypes, which possess differing sensitivities to the standard PCa therapies, such as androgen deprivation therapy (ADT). We argue that, although the structural plasticity of an IDP is important in promoting promiscuous interactions, the modulation of the structural ordering is important for sculpting its interactions so as to rewire with agility biomolecular interaction networks with significant functional consequences.

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Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy

10.26434/chemrxiv.11537913 ◽

2020 ◽

Author(s):

Franziska Zosel ◽

Andrea Holla ◽

Benjamin Schuler

Keyword(s):

Fluorescence Spectroscopy ◽

Single Molecule ◽

Intrinsically Disordered Proteins ◽

Conformational Dynamics ◽

Control Sample ◽

Resonance Energy ◽

Disordered Proteins ◽

Single Molecule Fluorescence ◽

Intrinsically Disordered ◽

Single Molecule Fluorescence Spectroscopy

Single-molecule fluorescence spectroscopy has become an important technique for studying the conformational dynamics and folding of proteins. A key step for performing such experiments is the availability of high-quality samples. Here we describe the practical details of a simple and widely applicable strategy for preparing proteins that are site-specifically labeled with a donor and an acceptor dye for single-molecule Förster resonance energy transfer (FRET) experiments. The method is based on introducing two cysteine residues that are labeled with maleimide-functionalized fluorophores, combined with high-resolution chromatography. We discuss how to optimize site-specific labeling even in the absence of orthogonal coupling chemistry and present purification strategies that are suitable for samples ranging from intrinsically disordered proteins to large folded proteins. We also discuss common problems in protein labeling, how to avoid them, and how to stringently control sample quality.<br>

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Hsp70 inhibits aggregation of Islet amyloid polypeptide by binding to the heterogeneous prenucleation oligomers

10.1101/2020.03.30.016881 ◽

2020 ◽

Author(s):

Neeraja Chilukoti ◽

Bankanidhi Sahoo ◽

S Deepa ◽

Sreelakshmi Cherakara ◽

Mithun Maddheshiya ◽

...

Keyword(s):

Intrinsically Disordered Proteins ◽

Islet Amyloid Polypeptide ◽

Correlation Spectroscopy ◽

Size Exclusion ◽

Disordered Proteins ◽

Strong Interactions ◽

Islet Amyloid ◽

Intrinsically Disordered ◽

Exclusion Chromatography ◽

In The Beginning

AbstractMolecular chaperone Hsp70 plays important roles in the pathology of amyloid diseases by inhibiting aberrant aggregation of proteins. However, mechanism of the interactions of Hsp70 with the amyloidogenic intrinsically disordered proteins (IDPs) is not clear. Here, we use Hsp70 from different organisms to show that it inhibits aggregation of Islet amyloid polypeptide (IAPP) at substoichiometric concentrations even in absence of ATP. The effect is found to be the strongest if Hsp70 is added in the beginning of aggregation but progressively less if added later, indicating role of Hsp70 in preventing primary nucleation possibly via interactions with the prefibrillar oligomers of IAPP. Fluorescence Correlation Spectroscopy (FCS) measurements of the solutions containing fluorescently labelled Hsp70 and IAPP exhibit fluorescence bursts suggesting formation of heterogeneous complexes of oligomeric IAPP binding to multiple molecules of Hsp70. Size exclusion chromatography and field flow fractionation are then used to fractionate the smaller complexes. Multiangle light scattering and FCS measurements suggest that these complexes comprise of monomers of Hsp70 and small oligomers of IAPP. However, concentration of the complexes is measured to be a few nanomolar amidst several μmolar of free Hsp70 and IAPP. Hence, our results indicate that Hsp70 interacts poorly with the monomers but strongly with oligomers of IAPP. This is likely a common feature of the interactions between the chaperones and the amyloidogenic IDPs. While strong interactions with the oligomers prevent aberrant aggregation, poor interaction with the monomers avert interference with the functions of the IDPs.

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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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Sequence-dependent correlated segments in the intrinsically disordered region of ChiZ

10.1101/2020.04.22.055590 ◽

2020 ◽

Author(s):

Alan Hicks ◽

Cristian A. Escobar ◽

Timothy A. Cross ◽

Huan-Xiang Zhou

Keyword(s):

Intrinsically Disordered Proteins ◽

Conformational Dynamics ◽

Md Simulations ◽

Proton Exchange ◽

Disordered Proteins ◽

Amide Proton ◽

Relaxation Rates ◽

Intrinsically Disordered ◽

Amide Proton Exchange ◽

Sequence Dependent

AbstractIntrinsically disordered proteins (IDPs) account for a significant fraction of any proteome and are central to numerous cellular functions. Yet how sequences of IDPs code for their conformational dynamics is poorly understood. Here we combined NMR spectroscopy, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations to characterize the conformations and dynamics of ChiZ1-64. This IDP is the N-terminal fragment (residues 1-64) of the transmembrane protein ChiZ, a component of the cell division machinery in Mycobacterium tuberculosis. Its N-half contains most of the prolines and all of the anionic residues while the C-half most of the glycines and cationic residues. MD simulations, first validated by SAXS and secondary chemical shift data, found scant α-helices or β-strands but considerable propensity for polyproline II (PPII) torsion angles. Importantly, several blocks of residues (e.g., 11-29) emerge as “correlated segments”, identified by frequent formation of PPII stretches, salt bridges, cation-π interactions, and sidechain-backbone hydrogen bonds. NMR relaxation experiments showed non-uniform transverse relaxation rates (R2s) and nuclear Overhauser enhancements (NOEs) along the sequence (e.g., high R2s and NOEs for residues 11-14 and 23-28). MD simulations further revealed that the extent of segmental correlation is sequence-dependent: segments where internal interactions are more prevalent manifest elevated “collective” motions on the 5-10 ns timescale and suppressed local motions on the sub-ns timescale. Amide proton exchange rates provides corroboration, with residues in the most correlated segment exhibiting the highest protection factors. We propose correlated segment as a defining feature for the conformation and dynamics of IDPs.

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Hierarchical Ensembles of Intrinsically Disordered Proteins at Atomic Resolution in Molecular Dynamics Simulations

10.1101/731133 ◽

2019 ◽

Author(s):

Lisa M. Pietrek ◽

Lukas S. Stelzl ◽

Gerhard Hummer

Keyword(s):

Molecular Dynamics ◽

Local Structure ◽

High Performance ◽

Intrinsically Disordered Proteins ◽

Large Fraction ◽

Conformational Dynamics ◽

Full Length ◽

Atomic Resolution ◽

Disordered Proteins ◽

Intrinsically Disordered

AbstractIntrinsically disordered proteins (IDPs) constitute a large fraction of the human proteome and are critical in the regulation of cellular processes. A detailed understanding of the conformational dynamics of IDPs could help to elucidate their roles in health and disease. However the inherent flexibility of IDPs makes structural studies and their interpretation challenging. Molecular dynamics (MD) simulations could address this challenge in principle, but inaccuracies in the simulation models and the need for long simulations have stymied progress. To overcome these limitations, we adopt an hierarchical approach that builds on the “flexible meccano” model of Bernadó et al. (J. Am. Chem. Soc. 2005, 127, 17968-17969). First, we exhaustively sample small IDP fragments in all-atom simulations to capture local structure. Then, we assemble the fragments into full-length IDPs to explore the stereochemically possible global structures of IDPs. The resulting ensembles of three-dimensional structures of full-length IDPs are highly diverse, much more so than in standard MD simulation. For the paradigmatic IDP α-synuclein, our ensemble captures both local structure, as probed by nuclear magnetic resonance (NMR) spectroscopy, and its overall dimension, as obtained from small-angle X-ray scattering (SAXS) in solution. By generating representative and meaningful starting ensembles, we can begin to exploit the massive parallelism afforded by current and future high-performance computing resources for atomic-resolution characterization of IDPs.

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Ensemble allosteric model: energetic frustration within the intrinsically disordered glucocorticoid receptor

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2017.0175 ◽

2018 ◽

Vol 373 (1749) ◽

pp. 20170175 ◽

Cited By ~ 10

Author(s):

Jordan T. White ◽

Jing Li ◽

Emily Grasso ◽

James O. Wrabl ◽

Vincent J. Hilser

Keyword(s):

Glucocorticoid Receptor ◽

Conformational Changes ◽

Intrinsically Disordered Proteins ◽

Disordered Proteins ◽

Coupling Energy ◽

Precise Control ◽

Intrinsically Disordered ◽

Protein Allostery ◽

Multiple Interfaces ◽

Allosteric Model

Allostery is an important regulatory phenomenon enabling precise control of biological function. Initial understanding of allostery was gained from seminal work on conformational changes exhibited by structured proteins. Within the last decade, protein allostery has also been demonstrated to occur within intrinsically disordered proteins. This emerging concept of disorder-mediated allostery can be usefully understood in the context of a thermodynamic ensemble. The advantage of this ensemble allosteric model is that it unifies the explanations of allostery occurring within both structured and disordered proteins. One central finding from this model is that energetic coupling, the transmission of a signal between separate regions (or domains) of a protein, is maximized when one or more domains are disordered. This is due to a disorder–order transition that contributes additional coupling energy to the allosteric system through formation of a molecular interaction surface or interface. A second key finding is that multiple interfaces may constructively or destructively interfere with each other, resulting in a new form of allosteric regulation called ‘energetic frustration’. Articulating protein allostery in terms of the thermodynamic ensemble permits formulation of experimentally testable hypotheses which can increase fundamental understanding and direct drug-design efforts. These ideas are illustrated here with the specific case of human glucocorticoid receptor, a medically important multi-domain allosteric protein that contains both structured and disordered regions and exemplifies ‘energetic frustration’. This article is part of a discussion meeting issue ‘Allostery and molecular machines’.

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