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

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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Determinants for Intrinsically Disordered Protein Recruitment into Phase-Separated Protein Condensates

Chemical Science ◽

10.1039/d1sc05672g ◽

2022 ◽

Author(s):

Yongsang Jo ◽

Jinyoung Jang ◽

Daesun Song ◽

Hyoin Park ◽

Yongwon Jung

Keyword(s):

Phase Separation ◽

Amino Acid ◽

Intrinsically Disordered Proteins ◽

Intrinsically Disordered Protein ◽

Disordered Proteins ◽

Amino Acid Residues ◽

Intrinsically Disordered ◽

Disordered Protein ◽

Protein Recruitment ◽

In Cells

Multivalent interactions between amino acid residues of intrinsically disordered proteins (IDPs) drive phase separation of these proteins into liquid condensates, forming various membrane-less organelles in cells. These interactions between often...

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Sequence fingerprints distinguish erroneous from correct predictions of Intrinsically Disordered Protein Regions

10.1101/203547 ◽

2017 ◽

Author(s):

Konda Mani Saravanan ◽

A Keith Dunker ◽

Sankaran Krishnaswamy

Keyword(s):

Amino Acid ◽

False Positive ◽

Intrinsically Disordered Proteins ◽

Intrinsically Disordered Protein ◽

Disordered Proteins ◽

Prediction Algorithm ◽

Amino Acid Residues ◽

Intrinsically Disordered ◽

Distinguishing Features

ABSTRACTMore than sixty prediction methods for intrinsically disordered proteins (IDPs) have been developed over the years, many of which are accessible on the world-wide web. Nearly, all of these predictors give balanced accuracies in the ~65% to ~80% range. Since predictors are not perfect, further studies are required to uncover the role of amino acid residues in native IDP as compared to predicted IDP regions. In the present work, we make use of sequences of 100% predicted IDP regions, false positive disorder predictions, and experimentally determined IDP regions to distinguish the characteristics of native versus predicted IDP regions. A higher occurrence of asparagine is observed in sequences of native IDP regions but not in sequences of false positive predictions of IDP regions. The occurrences of certain combinations of amino acids at the pentapeptide level provide a distinguishing feature in the IDPs with respect to globular proteins. The distinguishing features presented in this paper provide insights into the sequence fingerprints of amino acid residues in experimentally-determined as compared to predicted IDP regions. These observations and additional work along these lines should enable the development of improvements in the accuracy of disorder prediction algorithm.

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Mathematical and Computational Techniques for Drug Discovery: Promises and Developments

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666190208164005 ◽

2019 ◽

Vol 18 (32) ◽

pp. 2774-2799 ◽

Cited By ~ 8

Author(s):

Krishnan Balasubramanian

Keyword(s):

Drug Discovery ◽

Protein Interactions ◽

Quantum Chemical ◽

Intrinsically Disordered Proteins ◽

Hepatitis Virus ◽

Disordered Proteins ◽

Computational Techniques ◽

Large Set ◽

Structural Protein ◽

Intrinsically Disordered

We review various mathematical and computational techniques for drug discovery exemplifying some recent works pertinent to group theory of nested structures of relevance to phylogeny, topological, computational and combinatorial methods for drug discovery for multiple viral infections. We have reviewed techniques from topology, combinatorics, graph theory and knot theory that facilitate topological and mathematical characterizations of protein-protein interactions, molecular-target interactions, proteomics, genomics and statistical data reduction procedures for a large set of starting chemicals in drug discovery. We have provided an overview of group theoretical techniques pertinent to phylogeny, protein dynamics especially in intrinsically disordered proteins, DNA base permutations and related algorithms. We consider computational techniques derived from high level quantum chemical computations such as QM/MM ONIOM methods, quantum chemical optimization of geometries complexes, and molecular dynamics methods for providing insights into protein-drug interactions. We have considered complexes pertinent to Hepatitis Virus C non-structural protein 5B polymerase receptor binding of C5-Arylidebne rhodanines, complexes of synthetic potential vaccine molecules with dengue virus (DENV) and HIV-1 virus as examples of various simulation studies that exemplify the utility of computational tools. It is demonstrated that these combinatorial and computational techniques in conjunction with experiments can provide promising new insights into drug discovery. These techniques also demonstrate the need to consider a new multiple site or allosteric binding approach to drug discovery, as these studies reveal the existence of multiple binding sites.

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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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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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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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Sequence Versus Composition: What Prescribes IDP Biophysical Properties?

Entropy ◽

10.3390/e21070654 ◽

2019 ◽

Vol 21 (7) ◽

pp. 654 ◽

Cited By ~ 2

Author(s):

Jiří Vymětal ◽

Jiří Vondrášek ◽

Klára Hlouchová

Keyword(s):

Amino Acid ◽

Secondary Structure ◽

Intrinsically Disordered Proteins ◽

Random Sequence ◽

Bioinformatic Analysis ◽

Globular Proteins ◽

Disordered Proteins ◽

Compositional Bias ◽

Intrinsically Disordered ◽

Sequence Versus

Intrinsically disordered proteins (IDPs) represent a distinct class of proteins and are distinguished from globular proteins by conformational plasticity, high evolvability and a broad functional repertoire. Some of their properties are reminiscent of early proteins, but their abundance in eukaryotes, functional properties and compositional bias suggest that IDPs appeared at later evolutionary stages. The spectrum of IDP properties and their determinants are still not well defined. This study compares rudimentary physicochemical properties of IDPs and globular proteins using bioinformatic analysis on the level of their native sequences and random sequence permutations, addressing the contributions of composition versus sequence as determinants of the properties. IDPs have, on average, lower predicted secondary structure contents and aggregation propensities and biased amino acid compositions. However, our study shows that IDPs exhibit a broad range of these properties. Induced fold IDPs exhibit very similar compositions and secondary structure/aggregation propensities to globular proteins, and can be distinguished from unfoldable IDPs based on analysis of these sequence properties. While amino acid composition seems to be a major determinant of aggregation and secondary structure propensities, sequence randomization does not result in dramatic changes to these properties, but for both IDPs and globular proteins seems to fine-tune the tradeoff between folding and aggregation.

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Analysis of Heterodimeric “Mutual Synergistic Folding”-Complexes

International Journal of Molecular Sciences ◽

10.3390/ijms20205136 ◽

2019 ◽

Vol 20 (20) ◽

pp. 5136 ◽

Cited By ~ 3

Author(s):

Mentes ◽

Magyar ◽

Fichó ◽

Simon

Keyword(s):

Amino Acid ◽

Amino Acid Composition ◽

Acid Composition ◽

Intrinsically Disordered Proteins ◽

Driving Forces ◽

Disordered Proteins ◽

Subunit Interactions ◽

Amino Acid Compositions ◽

Intrinsically Disordered ◽

Β Sheet

Several intrinsically disordered proteins (IDPs) are capable to adopt stable structures without interacting with a folded partner. When the folding of all interacting partners happens at the same time, coupled with the interaction in a synergistic manner, the process is called Mutual Synergistic Folding (MSF). These complexes represent a discrete subset of IDPs. Recently, we collected information on their complexes and created the MFIB (Mutual Folding Induced by Binding) database. In a previous study, we compared homodimeric MSF complexes with homodimeric and monomeric globular proteins with similar amino acid sequence lengths. We concluded that MSF homodimers, compared to globular homodimeric proteins, have a greater solvent accessible main-chain surface area on the contact surface of the subunits, which becomes buried during dimerization. The main driving force of the folding is the mutual shielding of the water-accessible backbones, but the formation of further intermolecular interactions can also be relevant. In this paper, we will report analyses of heterodimeric MSF complexes. Our results indicate that the amino acid composition of the heterodimeric MSF monomer subunits slightly diverges from globular monomer proteins, while after dimerization, the amino acid composition of the overall MSF complexes becomes more similar to overall amino acid compositions of globular complexes. We found that inter-subunit interactions are strengthened, and additionally to the shielding of the solvent accessible backbone, other factors might play an important role in the stabilization of the heterodimeric structures, likewise energy gain resulting from the interaction of the two subunits with different amino acid compositions. We suggest that the shielding of the β-sheet backbones and the formation of a buried structural core along with the general strengthening of inter-subunit interactions together could be the driving forces of MSF protein structural ordering upon dimerization.

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