Protein conformation as a regulator of cell–matrix adhesion

Conformational changes within proteins play key roles in the regulation of cell–matrix adhesion. We discuss the mechanisms involved in conformational regulation, including mechanical signals, posttranslational modifications and intrinsically disordered proteins.

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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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Structural Proteomics Methods to Interrogate the Conformations and Dynamics of Intrinsically Disordered Proteins

Frontiers in Chemistry ◽

10.3389/fchem.2021.603639 ◽

2021 ◽

Vol 9 ◽

Author(s):

Rebecca Beveridge ◽

Antonio N. Calabrese

Keyword(s):

Posttranslational Modifications ◽

Intrinsically Disordered Proteins ◽

Structure And Function ◽

Disordered Proteins ◽

Structural Proteomics ◽

Intrinsically Disordered ◽

Folded Proteins ◽

Structural Mass Spectrometry ◽

And Function ◽

Structural Mass

Intrinsically disordered proteins (IDPs) and regions of intrinsic disorder (IDRs) are abundant in proteomes and are essential for many biological processes. Thus, they are often implicated in disease mechanisms, including neurodegeneration and cancer. The flexible nature of IDPs and IDRs provides many advantages, including (but not limited to) overcoming steric restrictions in binding, facilitating posttranslational modifications, and achieving high binding specificity with low affinity. IDPs adopt a heterogeneous structural ensemble, in contrast to typical folded proteins, making it challenging to interrogate their structure using conventional tools. Structural mass spectrometry (MS) methods are playing an increasingly important role in characterizing the structure and function of IDPs and IDRs, enabled by advances in the design of instrumentation and the development of new workflows, including in native MS, ion mobility MS, top-down MS, hydrogen-deuterium exchange MS, crosslinking MS, and covalent labeling. Here, we describe the advantages of these methods that make them ideal to study IDPs and highlight recent applications where these tools have underpinned new insights into IDP structure and function that would be difficult to elucidate using other methods.

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

10.1101/2022.01.14.476386 ◽

2022 ◽

Author(s):

Spencer Smyth ◽

Zhenfu Zhang ◽

Alaji Bah ◽

Thomas Tsangaris ◽

Jennifer Dawson ◽

...

Keyword(s):

Conformational Changes ◽

Intrinsically Disordered Proteins ◽

Regulatory Protein ◽

Conformational Dynamics ◽

Correlation Spectroscopy ◽

Terminal Region ◽

Disordered Proteins ◽

Initiation Factor ◽

Dependent Manner ◽

Intrinsically Disordered

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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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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Combinatorial phosphorylation modulates the structure and function of the G protein γ subunit in yeast

Science Signaling ◽

10.1126/scisignal.abd2464 ◽

2021 ◽

Vol 14 (688) ◽

pp. eabd2464

Author(s):

Zahra Nassiri Toosi ◽

Xinya Su ◽

Ruth Austin ◽

Shilpa Choudhury ◽

Wei Li ◽

...

Keyword(s):

G Protein ◽

Posttranslational Modifications ◽

Intrinsically Disordered Proteins ◽

Structure And Function ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Gpcr Activation ◽

Essential Components ◽

And Function

Intrinsically disordered regions (IDRs) in proteins are often targets of combinatorial posttranslational modifications, which serve to regulate protein structure and function. Emerging evidence suggests that the N-terminal tails of G protein γ subunits, which are essential components of heterotrimeric G proteins, are intrinsically disordered, phosphorylation-dependent determinants of G protein signaling. Here, we found that the yeast Gγ subunit Ste18 underwent combinatorial, multisite phosphorylation events within its N-terminal IDR. G protein–coupled receptor (GPCR) activation and osmotic stress induced phosphorylation at Ser7, whereas glucose and acid stress induced phosphorylation at Ser3, which was a quantitative indicator of intracellular pH. Each site was phosphorylated by a distinct set of kinases, and phosphorylation of one site affected phosphorylation of the other, as determined through exposure to serial stimuli and through phosphosite mutagenesis. Last, we showed that phosphorylation resulted in changes in IDR structure and that different combinations of phosphorylation events modulated the activation rate and amplitude of the downstream mitogen-activated protein kinase Fus3. These data place Gγ subunits among intrinsically disordered proteins that undergo combinatorial posttranslational modifications that govern signaling pathway output.

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Depletion interactions modulate the binding between disordered proteins in crowded environments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1921617117 ◽

2020 ◽

Vol 117 (24) ◽

pp. 13480-13489 ◽

Cited By ~ 11

Author(s):

Franziska Zosel ◽

Andrea Soranno ◽

Karin J. Buholzer ◽

Daniel Nettels ◽

Benjamin Schuler

Keyword(s):

Single Molecule ◽

Posttranslational Modifications ◽

Intrinsically Disordered Proteins ◽

Disordered Proteins ◽

Dissociation Kinetics ◽

Cellular Targets ◽

Intrinsically Disordered ◽

Functional Consequences ◽

Folded Proteins ◽

Depletion Interactions

Intrinsically disordered proteins (IDPs) abound in cellular regulation. Their interactions are often transitory and highly sensitive to salt concentration and posttranslational modifications. However, little is known about the effect of macromolecular crowding on the interactions of IDPs with their cellular targets. Here, we investigate the influence of crowding on the interaction between two IDPs that fold upon binding, with polyethylene glycol as a crowding agent. Single-molecule spectroscopy allows us to quantify the effects of crowding on a comprehensive set of observables simultaneously: the equilibrium stability of the complex, the association and dissociation kinetics, and the microviscosity, which governs translational diffusion. We show that a quantitative and coherent explanation of all observables is possible within the framework of depletion interactions if the polymeric nature of IDPs and crowders is incorporated based on recent theoretical developments. The resulting integrated framework can also rationalize important functional consequences, for example, that the interaction between the two IDPs is less enhanced by crowding than expected for folded proteins of the same size.

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Energetics and kinetics of substrate analog-coupled staphylococcal nuclease folding revealed by a statistical mechanical approach

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914349117 ◽

2020 ◽

Vol 117 (33) ◽

pp. 19953-19962

Author(s):

Takuya Mizukami ◽

Shunta Furuzawa ◽

Satoru G. Itoh ◽

Saho Segawa ◽

Teikichi Ikura ◽

...

Keyword(s):

Conformational Changes ◽

Intrinsically Disordered Proteins ◽

Flux Ratio ◽

Staphylococcal Nuclease ◽

Binding Energies ◽

Disordered Proteins ◽

Flux Analysis ◽

Intrinsically Disordered ◽

Substrate Analog ◽

Statistical Mechanical

Protein conformational changes associated with ligand binding, especially those involving intrinsically disordered proteins, are mediated by tightly coupled intra- and intermolecular events. Such reactions are often discussed in terms of two limiting kinetic mechanisms, conformational selection (CS), where folding precedes binding, and induced fit (IF), where binding precedes folding. It has been shown that coupled folding/binding reactions can proceed along both CS and IF pathways with the flux ratio depending on conditions such as ligand concentration. However, the structural and energetic basis of such complex reactions remains poorly understood. Therefore, we used experimental, theoretical, and computational approaches to explore structural and energetic aspects of the coupled-folding/binding reaction of staphylococcal nuclease in the presence of the substrate analog adenosine-3′,5′-diphosphate. Optically monitored equilibrium and kinetic data, combined with a statistical mechanical model, gave deeper insight into the relative importance of specific and Coulombic protein–ligand interactions in governing the reaction mechanism. We also investigated structural aspects of the reaction at the residue level using NMR and all-atom replica-permutation molecular dynamics simulations. Both approaches yielded clear evidence for accumulation of a transient protein–ligand encounter complex early in the reaction under IF-dominant conditions. Quantitative analysis of the equilibrium/kinetic folding revealed that the ligand-dependent CS-to-IF shift resulted from stabilization of the compact transition state primarily by weakly ligand-dependent Coulombic interactions with smaller contributions from specific binding energies. At a more macroscopic level, the CS-to-IF shift was represented as a displacement of the reaction “route” on the free energy surface, which was consistent with a flux analysis.

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Depicting Conformational Ensembles of α-Synuclein by Single Molecule Force Spectroscopy and Native Mass Spectroscopy

International Journal of Molecular Sciences ◽

10.3390/ijms20205181 ◽

2019 ◽

Vol 20 (20) ◽

pp. 5181 ◽

Cited By ~ 2

Author(s):

Roberta Corti ◽

Claudia A. Marrano ◽

Domenico Salerno ◽

Stefania Brocca ◽

Antonino Natalello ◽

...

Keyword(s):

Mass Spectrometry ◽

Single Molecule ◽

Conformational Changes ◽

Intrinsically Disordered Proteins ◽

Force Spectroscopy ◽

Native Mass Spectrometry ◽

Disordered Proteins ◽

Unique Contribution ◽

Single Molecule Force Spectroscopy ◽

Intrinsically Disordered

Description of heterogeneous molecular ensembles, such as intrinsically disordered proteins, represents a challenge in structural biology and an urgent question posed by biochemistry to interpret many physiologically important, regulatory mechanisms. Single-molecule techniques can provide a unique contribution to this field. This work applies single molecule force spectroscopy to probe conformational properties of α-synuclein in solution and its conformational changes induced by ligand binding. The goal is to compare data from such an approach with those obtained by native mass spectrometry. These two orthogonal, biophysical methods are found to deliver a complex picture, in which monomeric α-synuclein in solution spontaneously populates compact and partially compacted states, which are differently stabilized by binding to aggregation inhibitors, such as dopamine and epigallocatechin-3-gallate. Analyses by circular dichroism and Fourier-transform infrared spectroscopy show that these transitions do not involve formation of secondary structure. This comparative analysis provides support to structural interpretation of charge-state distributions obtained by native mass spectrometry and helps, in turn, defining the conformational components detected by single molecule force spectroscopy.

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