scholarly journals Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity

2017 ◽  
Vol 114 (13) ◽  
pp. E2644-E2653 ◽  
Author(s):  
Prakash Kulkarni ◽  
Mohit Kumar Jolly ◽  
Dongya Jia ◽  
Steven M. Mooney ◽  
Ajay Bhargava ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Large Fraction ◽  
Conformational Dynamics ◽  
3D Structure ◽  
Random Coil ◽  
Disordered Proteins ◽  
Conformational Ensemble ◽  
Intrinsically Disordered ◽  
Androgen Independent

Intrinsically disordered proteins (IDPs) that lack a unique 3D structure and comprise a large fraction of the human proteome play important roles in numerous cellular functions. Prostate-Associated Gene 4 (PAGE4) is an IDP that acts as a potentiator of the Activator Protein-1 (AP-1) transcription factor. Homeodomain-Interacting Protein Kinase 1 (HIPK1) phosphorylates PAGE4 at S9 and T51, but only T51 is critical for its activity. Here, we identify a second kinase, CDC-Like Kinase 2 (CLK2), which acts on PAGE4 and hyperphosphorylates it at multiple S/T residues, including S9 and T51. We demonstrate that HIPK1 is expressed in both androgen-dependent and androgen-independent prostate cancer (PCa) cells, whereas CLK2 and PAGE4 are expressed only in androgen-dependent cells. Cell-based studies indicate that PAGE4 interaction with the two kinases leads to opposing functions. HIPK1-phosphorylated PAGE4 (HIPK1-PAGE4) potentiates c-Jun, whereas CLK2-phosphorylated PAGE4 (CLK2-PAGE4) attenuates c-Jun activity. Consistent with the cellular data, biophysical measurements (small-angle X-ray scattering, single-molecule fluorescence resonance energy transfer, and NMR) indicate that HIPK1-PAGE4 exhibits a relatively compact conformational ensemble that binds AP-1, whereas CLK2-PAGE4 is more expanded and resembles a random coil with diminished affinity for AP-1. Taken together, the results suggest that the phosphorylation-induced conformational dynamics of PAGE4 may play a role in modulating changes between PCa cell phenotypes. A mathematical model based on our experimental data demonstrates how differential phosphorylation of PAGE4 can lead to transitions between androgen-dependent and androgen-independent phenotypes by altering the AP-1/androgen receptor regulatory circuit in PCa cells.

scholarly journals Recent Advances in Computational Protocols Addressing Intrinsically Disordered Proteins

Biomolecules ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 146 ◽  
Author(s):  
Supriyo Bhattacharya ◽  
Xingcheng Lin
Keyword(s):  
Single Molecule ◽  
Degrees Of Freedom ◽  
Intrinsically Disordered Proteins ◽  
Structural Information ◽  
Conformational Dynamics ◽  
Resonance Energy ◽  
Structural Data ◽  
Disordered Proteins ◽  
Conformational Ensemble ◽  
Intrinsically Disordered

Intrinsically disordered proteins (IDP) are abundant in the human genome and have recently emerged as major therapeutic targets for various diseases. Unlike traditional proteins that adopt a definitive structure, IDPs in free solution are disordered and exist as an ensemble of conformations. This enables the IDPs to signal through multiple signaling pathways and serve as scaffolds for multi-protein complexes. The challenge in studying IDPs experimentally stems from their disordered nature. Nuclear magnetic resonance (NMR), circular dichroism, small angle X-ray scattering, and single molecule Förster resonance energy transfer (FRET) can give the local structural information and overall dimension of IDPs, but seldom provide a unified picture of the whole protein. To understand the conformational dynamics of IDPs and how their structural ensembles recognize multiple binding partners and small molecule inhibitors, knowledge-based and physics-based sampling techniques are utilized in-silico, guided by experimental structural data. However, efficient sampling of the IDP conformational ensemble requires traversing the numerous degrees of freedom in the IDP energy landscape, as well as force-fields that accurately model the protein and solvent interactions. In this review, we have provided an overview of the current state of computational methods for studying IDP structure and dynamics and discussed the major challenges faced in this field.

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

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.

scholarly journals Intrinsically Disordered Proteins in Chronic Diseases

Biomolecules ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 147 ◽  
Author(s):  
Prakash Kulkarni ◽  
Vladimir Uversky
Keyword(s):  
Chronic Diseases ◽  
Intrinsically Disordered Proteins ◽  
Large Fraction ◽  
3D Structure ◽  
Human Proteome ◽  
Disordered Proteins ◽  
3D Structures ◽  
Physiological Conditions ◽  
Intrinsically Disordered

It is now increasingly evident that a large fraction of the human proteome comprises proteins that, under physiological conditions, lack fixed, ordered 3D structures as a whole or have segments that are not likely to form a defined 3D structure [...]

scholarly journals Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy

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>

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

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.

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

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.

scholarly journals Characterization of Weak Protein Domain Structure by Spin-Label Distance Distributions

2021 ◽  
Vol 8 ◽  
Author(s):  
Irina Ritsch ◽  
Laura Esteban-Hofer ◽  
Elisabeth Lehmann ◽  
Leonidas Emmanouilidis ◽  
Maxim Yulikov ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Distance Distribution ◽  
Protein Domain ◽  
Random Coil ◽  
Disordered Proteins ◽  
Primary Data ◽  
Spin Labels ◽  
Conformational Ensemble ◽  
Intrinsically Disordered ◽  
Distance Distributions

Function of intrinsically disordered proteins may depend on deviation of their conformational ensemble from that of a random coil. Such deviation may be hard to characterize and quantify, if it is weak. We explored the potential of distance distributions between spin labels, as they can be measured by electron paramagnetic resonance techniques, for aiding such characterization. On the example of the intrinsically disordered N-terminal domain 1–267 of fused in sarcoma (FUS) we examined what such distance distributions can and cannot reveal on the random-coil reference state. On the example of the glycine-rich domain 188–320 of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) we studied whether deviation from a random-coil ensemble can be robustly detected with 19 distance distribution restraints. We discuss limitations imposed by ill-posedness of the conversion of primary data to distance distributions and propose overlap of distance distributions as a fit criterion that can tackle this problem. For testing consistency and size sufficiency of the restraint set, we propose jack-knife resampling. At current desktop computers, our approach is expected to be viable for domains up to 150 residues and for between 10 and 50 distance distribution restraints.

scholarly journals Dual roles of electrostatic-steering and conformational dynamics in the binding of calcineurin’s intrinsically-disordered recognition domain to calmodulin

2018 ◽  
Author(s):  
Bin Sun ◽  
Eric C. Cook ◽  
Trevor P. Creamer ◽  
Peter M. Kekenes-Huskey
Keyword(s):  
Long Range ◽  
Electrostatic Interactions ◽  
Intrinsically Disordered Proteins ◽  
Conformational Dynamics ◽  
Disordered Proteins ◽  
Conformational Ensemble ◽  
Regulatory Domain ◽  
Intrinsically Disordered ◽  
Diffusion Limited ◽  
Conformational Diversity

calcineurin (CaN) is a serine/threonine phosphatase that regulates a variety of physiological and pathophysiological processes in mammalian tissue. The CaN regulatory domain (RD) is responsible for regulating the enzyme’s phosphatase activity, and is believed to be highly-disordered when inhibiting CaN, but undergoes a disorderto-order transition upon diffusion-limited binding with the regulatory protein calmodulin (CaM). The prevalence of polar and charged amino acids in the regulatory domain (RD) suggests electrostatic interactions are involved in mediating CaM binding, yet the lack of atomistic-resolution data for the bound complex has stymied efforts to probe how the RD sequence controls its conformational ensemble and long-range attractions contribute to target protein binding. In the present study, we investigated via computational modeling the extent to which electrostatics and structural disorder cofacilitate or hinder CaM/CaN association kinetics. Specifically, we examined several RD constructs that contain the CaM binding region (CAMBR) to characterize the roles of electrostatics versus conformational diversity in controlling diffusion-limited association rates, via microsecond-scale molecular dynamics (MD) and Brownian dynamic (BD) simulations. Our results indicate that the RD amino acid composition and sequence length influence both the dynamic availability of conformations amenable to CaM binding, as well as long-range electrostatic interactions to steer association. These findings provide intriguing insight into the interplay between conformational diversity and electrostatically-driven protein-protein association involving CaN, which are likely to extend to wide-ranging diffusion-limited processes regulated by intrinsically-disordered proteins.

scholarly journals Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy

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>

scholarly journals Extreme Fuzziness: Direct Interactions between Two IDPs

Biomolecules ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 81 ◽  
Author(s):  
Wenning Wang ◽  
Dongdong Wang
Keyword(s):  
Single Molecule ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Intrinsically Disordered Proteins ◽  
Conformational Dynamics ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Full Spectrum ◽  
Intrinsically Disordered ◽  
Binding Mechanisms

Protein interactions involving intrinsically disordered proteins (IDPs) greatly extend the range of binding mechanisms available to proteins. In interactions employing coupled folding and binding, IDPs undergo disorder-to-order transitions to form a complex with a well-defined structure. In many other cases, IDPs retain structural plasticity in the final complexes, which have been defined as the fuzzy complexes. While a large number of fuzzy complexes have been characterized with variety of fuzzy patterns, many of the interactions are between an IDP and a structured protein. Thus, whether two IDPs can interact directly to form a fuzzy complex without disorder-to-order transition remains an open question. Recently, two studies of interactions between IDPs (4.1G-CTD/NuMA and H1/ProTα) have found a definite answer to this question. Detailed characterizations combined with nuclear magnetic resonance (NMR), single-molecule Förster resonance energy transfer (smFRET) and molecular dynamics (MD) simulation demonstrate that direct interactions between these two pairs of IDPs do form fuzzy complexes while retaining the conformational dynamics of the isolated proteins, which we name as the extremely fuzzy complexes. Extreme fuzziness completes the full spectrum of protein-protein interaction modes, suggesting that a more generalized model beyond existing binding mechanisms is required. Previous models of protein interaction could be applicable to some aspects of the extremely fuzzy interactions, but in more general sense, the distinction between native and nonnative contacts, which was used to understand protein folding and binding, becomes obscure. Exploring the phenomenon of extreme fuzziness may shed new light on molecular recognition and drug design.

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