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1.
Int J Mol Sci ; 23(7)2022 Mar 31.
Article in English | MEDLINE | ID: covidwho-1776247

ABSTRACT

In a recent paper, we proposed the folding interdiction target region (FITR) strategy for therapeutic drug design in SARS-CoV-2. This paper expands the application of the FITR strategy by proposing therapeutic drug design approaches against Ebola virus disease and influenza A. We predict target regions for folding interdicting drugs on correspondingly relevant structural proteins of both pathogenic viruses: VP40 of Ebola, and matrix protein M1 of influenza A. Identification of the protein targets employs the sequential collapse model (SCM) for protein folding. It is explained that the model predicts natural peptide candidates in each case from which to start the search for therapeutic drugs. The paper also discusses how these predictions could be tested, as well as some challenges likely to be found when designing effective therapeutic drugs from the proposed peptide candidates. The FITR strategy opens a potential new avenue for the design of therapeutic drugs that promises to be effective against infectious diseases.


Subject(s)
COVID-19 , Ebolavirus , Hemorrhagic Fever, Ebola , Influenza, Human , Drug Development , Ebolavirus/metabolism , Hemorrhagic Fever, Ebola/drug therapy , Humans , Influenza, Human/drug therapy , Protein Folding , SARS-CoV-2 , Viral Matrix Proteins/metabolism
2.
PLoS Comput Biol ; 18(2): e1009833, 2022 02.
Article in English | MEDLINE | ID: covidwho-1731576

ABSTRACT

As sequence and structure comparison algorithms gain sensitivity, the intrinsic interconnectedness of the protein universe has become increasingly apparent. Despite this general trend, ß-trefoils have emerged as an uncommon counterexample: They are an isolated protein lineage for which few, if any, sequence or structure associations to other lineages have been identified. If ß-trefoils are, in fact, remote islands in sequence-structure space, it implies that the oligomerizing peptide that founded the ß-trefoil lineage itself arose de novo. To better understand ß-trefoil evolution, and to probe the limits of fragment sharing across the protein universe, we identified both 'ß-trefoil bridging themes' (evolutionarily-related sequence segments) and 'ß-trefoil-like motifs' (structure motifs with a hallmark feature of the ß-trefoil architecture) in multiple, ostensibly unrelated, protein lineages. The success of the present approach stems, in part, from considering ß-trefoil sequence segments or structure motifs rather than the ß-trefoil architecture as a whole, as has been done previously. The newly uncovered inter-lineage connections presented here suggest a novel hypothesis about the origins of the ß-trefoil fold itself-namely, that it is a derived fold formed by 'budding' from an Immunoglobulin-like ß-sandwich protein. These results demonstrate how the evolution of a folded domain from a peptide need not be a signature of antiquity and underpin an emerging truth: few protein lineages escape nature's sewing table.


Subject(s)
Lotus , Immunoglobulin G , Models, Molecular , Peptides/chemistry , Protein Folding
3.
Int J Biol Macromol ; 197: 68-76, 2022 Feb 01.
Article in English | MEDLINE | ID: covidwho-1587673

ABSTRACT

The C-terminal domain of SARS-CoV main protease (Mpro-C) can form 3D domain-swapped dimer by exchanging the α1-helices fully buried inside the protein hydrophobic core, under non-denaturing conditions. Here, we report that Mpro-C can also form amyloid fibrils under the 3D domain-swappable conditions in vitro, and the fibrils are not formed through runaway/propagated domain swapping. It is found that there are positive correlations between the rates of domain swapping dimerization and amyloid fibrillation at different temperatures, and for different mutants. However, some Mpro-C mutants incapable of 3D domain swapping can still form amyloid fibrils, indicating that 3D domain swapping is not essential for amyloid fibrillation. Furthermore, NMR H/D exchange data and molecular dynamics simulation results suggest that the protofibril core region tends to unpack at the early stage of 3D domain swapping, so that the amyloid fibrillation can proceed during the 3D domain swapping process. We propose that 3D domain swapping makes it possible for the unpacking of the amyloidogenic fragment of the protein and thus accelerates the amyloid fibrillation process kinetically, which explains the well-documented correlations between amyloid fibrillation and 3D domain swapping observed in many proteins.


Subject(s)
Amyloid/chemistry , Amyloid/metabolism , Amyloidosis/metabolism , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Protein Domains/physiology , Amyloidosis/genetics , Coronavirus 3C Proteases/genetics , Dimerization , Disulfides/chemistry , Disulfides/metabolism , Kinetics , Models, Molecular , Molecular Dynamics Simulation , Mutation , Polymerization , Protein Conformation, alpha-Helical , Protein Domains/genetics , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Temperature
4.
Nat Struct Mol Biol ; 28(7): 614-625, 2021 07.
Article in English | MEDLINE | ID: covidwho-1550333

ABSTRACT

p97 processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Here, we report a series of cryo-EM structures of the substrate-engaged human p97 complex with resolutions ranging from 2.9 to 3.8 Å that captured 'power-stroke'-like motions of both the D1 and D2 ATPase rings of p97. A key feature of these structures is the critical conformational changes of the intersubunit signaling (ISS) motifs, which tighten the binding of nucleotides and neighboring subunits and contribute to the spiral staircase conformation of the D1 and D2 rings. In addition, we determined the cryo-EM structure of human p97 in complex with NMS-873, a potent p97 inhibitor, at a resolution of 2.4 Å. The structures showed that NMS-873 binds at a cryptic groove in the D2 domain and interacts with the ISS motif, preventing its conformational change and thus blocking substrate translocation allosterically.


Subject(s)
Adenosine Triphosphate/chemistry , Protein Folding , Proteostasis/physiology , Signal Transduction/physiology , Valosin Containing Protein/metabolism , Acetanilides/pharmacology , Animals , Benzothiazoles/pharmacology , Cryoelectron Microscopy , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum-Associated Degradation/physiology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Conformation , Ubiquitinated Proteins/metabolism , Valosin Containing Protein/antagonists & inhibitors
5.
Biomolecules ; 11(12)2021 12 02.
Article in English | MEDLINE | ID: covidwho-1551563

ABSTRACT

COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful "tool" to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.


Subject(s)
COVID-19/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Animals , Cell Line , Escherichia coli/genetics , Gene Expression , HEK293 Cells , Humans , Insecta/cytology , Protein Binding , Protein Denaturation , Protein Domains , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
6.
Protein Sci ; 31(1): 283-289, 2022 01.
Article in English | MEDLINE | ID: covidwho-1516798

ABSTRACT

The PDBsum web server provides structural analyses of the entries in the Protein Data Bank (PDB). Two recent additions are described here. The first is the detailed analysis of the SARS-CoV-2 virus protein structures in the PDB. These include the variants of concern, which are shown both on the sequences and 3D structures of the proteins. The second addition is the inclusion of the available AlphaFold models for human proteins. The pages allow a search of the protein against existing structures in the PDB via the Sequence Annotated by Structure (SAS) server, so one can easily compare the predicted model against experimentally determined structures. The server is freely accessible to all at http://www.ebi.ac.uk/pdbsum.


Subject(s)
Databases, Protein , Proteins/chemistry , SARS-CoV-2/chemistry , Viral Proteins/chemistry , Animals , COVID-19/virology , Humans , Models, Molecular , Protein Conformation , Protein Folding , Software
7.
J Am Chem Soc ; 143(46): 19306-19310, 2021 11 24.
Article in English | MEDLINE | ID: covidwho-1510556

ABSTRACT

The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, Mpro (3CLpro/Nsp5), is a promising antiviral drug target. We evaluate the concordance of models generated by the newly introduced AlphaFold2 structure prediction program with residual dipolar couplings (RDCs) measured in solution for 15N-1HN and 13C'-1HN atom pairs. The latter were measured using a new, highly precise TROSY-AntiTROSY Encoded RDC (TATER) experiment. Three sets of AlphaFold2 models were evaluated: (1) MproAF, generated using the standard AlphaFold2 input structural database; (2) MproAFD, where the AlphaFold2 implementation was modified to exclude all candidate template X-ray structures deposited after Jan 1, 2020; and (3) MproAFS, which excluded all structures homologous to coronaviral Mpro. Close agreement between all three sets of AlphaFold models and experimental RDC data is found for most of the protein. For residues in well-defined secondary structure, the agreement decreases somewhat upon Amber relaxation. For these regions, MproAF agreement exceeds that of most high-resolution X-ray structures. Residues from domain 2 that comprise elements of both the active site and the homo-dimerization interface fit less well across all structures. These results indicate novel opportunities for combining experimentation with molecular dynamics simulations, where solution RDCs provide highly precise input for QM/MM simulations of substrate binding/reaction trajectories.


Subject(s)
Coronavirus 3C Proteases/chemistry , Crystallography, X-Ray/methods , SARS-CoV-2 , COVID-19 , Catalytic Domain , Magnetic Resonance Spectroscopy , Molecular Conformation , Molecular Dynamics Simulation , Protein Conformation , Protein Folding , Software , X-Rays
8.
Sci Rep ; 11(1): 22042, 2021 11 11.
Article in English | MEDLINE | ID: covidwho-1510622

ABSTRACT

The mutation of SARS-CoV-2 influences viral function as residue replacements affect both physiochemical properties and folding conformations. Although a large amount of data on SARS-CoV-2 is available, the investigation of how viral functions change in response to mutations is hampered by a lack of effective structural analysis. Here, we exploit the advances of protein structure fingerprint technology to study the folding conformational changes induced by mutations. With integration of both protein sequences and folding conformations, the structures are aligned for SARS-CoV to SARS-CoV-2, including Alpha variant (lineage B.1.1.7) and Delta variant (lineage B.1.617.2). The results showed that the virus evolution with change in mutational positions and physicochemical properties increased the affinity between spike protein and ACE2, which plays a critical role in coronavirus entry into human cells. Additionally, these structural variations impact vaccine effectiveness and drug function over the course of SARS-CoV-2 evolution. The analysis of structural variations revealed how the coronavirus has gradually evolved in both structure and function and how the SARS-CoV-2 variants have contributed to more severe acute disease worldwide.


Subject(s)
COVID-19/virology , Evolution, Molecular , Mutation , SARS-CoV-2/genetics , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Humans , Models, Molecular , Protein Conformation , Protein Folding , Protein Interaction Maps , Protein Multimerization , SARS Virus/chemistry , SARS Virus/genetics , SARS Virus/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
9.
Science ; 374(6575): 1621-1626, 2021 Dec 24.
Article in English | MEDLINE | ID: covidwho-1506414

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission leads to the emergence of variants, including the B.1.617.2 (Delta) variant of concern that is causing a new wave of infections and has become globally dominant. We show that these variants dampen the in vitro potency of vaccine-elicited serum neutralizing antibodies and provide a structural framework for describing their immune evasion. Mutations in the B.1.617.1 (Kappa) and Delta spike glycoproteins abrogate recognition by several monoclonal antibodies via alteration of key antigenic sites, including remodeling of the Delta amino-terminal domain. The angiotensin-converting enzyme 2 binding affinities of the Kappa and Delta receptor binding domains are comparable to the Wuhan-Hu-1 isolate, whereas B.1.617.2+ (Delta+) exhibits markedly reduced affinity.


Subject(s)
COVID-19 Vaccines/immunology , Immune Evasion , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , /immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , Antigens, Viral/chemistry , Antigens, Viral/immunology , Cryoelectron Microscopy , Humans , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Protein Domains , Protein Folding , Receptors, Coronavirus/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
10.
Virus Res ; 307: 198618, 2022 01 02.
Article in English | MEDLINE | ID: covidwho-1504602

ABSTRACT

The second wave of COVID-19 caused by severe acute respiratory syndrome virus (SARS-CoV-2) is rapidly spreading over the world. Mechanisms behind the flee from current antivirals are still unclear due to the continuous occurrence of SARS-CoV-2 genetic variants. Brazil is the world's second-most COVID-19 affected country. In the present study, we identified the genomic and proteomic variants of Brazilian SARS-CoV-2 isolates. We identified 16 different genotypic variants were found among the 27 isolates. The genotypes of three isolates such as Bra/1236/2021 (G15), Bra/MASP2C844R2/2020 (G11), and Bra/RJ-DCVN5/2020 (G9) have a unique mutant in NSP4 (S184N), 2'O-Mutase (R216N), membrane protein (A2V) and Envelope protein (V5A). A mutation in RdRp of SARS-CoV-2, particularly the change of Pro-to Leu-at 323 resulted in the stabilization of the structure in BRA/CD1739-P4/2020. NSP4, NSP5 protein mutants are more virulent in genotype 15 and 16. A fast protein folding rate changes the structural stability and leads to escape for current antivirals. Thus, our findings help researchers to develop the best potent antivirals based on the new mutant of Brazilian isolates.


Subject(s)
Coronavirus 3C Proteases/genetics , Protein Folding , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Brazil , COVID-19/pathology , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/genetics , Genetic Variation/genetics , Genome, Viral/genetics , Humans , Phosphoproteins/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Virulence/genetics
11.
Commun Biol ; 4(1): 1240, 2021 10 29.
Article in English | MEDLINE | ID: covidwho-1493232

ABSTRACT

Circular tandem repeat proteins ('cTRPs') are de novo designed protein scaffolds (in this and prior studies, based on antiparallel two-helix bundles) that contain repeated protein sequences and structural motifs and form closed circular structures. They can display significant stability and solubility, a wide range of sizes, and are useful as protein display particles for biotechnology applications. However, cTRPs also demonstrate inefficient self-assembly from smaller subunits. In this study, we describe a new generation of cTRPs, with longer repeats and increased interaction surfaces, which enhanced the self-assembly of two significantly different sizes of homotrimeric constructs. Finally, we demonstrated functionalization of these constructs with (1) a hexameric array of peptide-binding SH2 domains, and (2) a trimeric array of anti-SARS CoV-2 VHH domains. The latter proved capable of sub-nanomolar binding affinities towards the viral receptor binding domain and potent viral neutralization function.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Protein Engineering/methods , Proteins/chemistry , Proteins/metabolism , SARS-CoV-2/metabolism , Tandem Repeat Sequences , Amino Acid Sequence , COVID-19/virology , Computer Simulation , Crystallization , HEK293 Cells , Humans , Models, Molecular , Neutralization Tests , Protein Binding , Protein Domains , Protein Folding , Protein Structure, Secondary , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
12.
Nat Commun ; 12(1): 5739, 2021 10 19.
Article in English | MEDLINE | ID: covidwho-1475293

ABSTRACT

Protein aggregates associated with neurodegenerative diseases have the ability to transmit to unaffected cells, thereby templating their own aberrant conformation onto soluble homotypic proteins. Proteopathic seeds can be released into the extracellular space, secreted in association with extracellular vesicles (EV) or exchanged by direct cell-to-cell contact. The extent to which each of these pathways contribute to the prion-like spreading of protein misfolding is unclear. Exchange of cellular cargo by both direct cell contact or via EV depends on receptor-ligand interactions. We hypothesized that enabling these interactions through viral ligands enhances intercellular proteopathic seed transmission. Using different cellular models propagating prions or pathogenic Tau aggregates, we demonstrate that vesicular stomatitis virus glycoprotein and SARS-CoV-2 spike S increase aggregate induction by cell contact or ligand-decorated EV. Thus, receptor-ligand interactions are important determinants of intercellular aggregate dissemination. Our data raise the possibility that viral infections contribute to proteopathic seed spreading by facilitating intercellular cargo transfer.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Extracellular Vesicles/metabolism , Membrane Glycoproteins/metabolism , Protein Aggregation, Pathological/virology , Spike Glycoprotein, Coronavirus/metabolism , Viral Envelope Proteins/metabolism , Adult , Aged , Brain/pathology , Case-Control Studies , Cell Line , Endocytosis , Female , Humans , Intravital Microscopy , Male , Middle Aged , Prions/metabolism , Protein Aggregation, Pathological/pathology , Protein Folding , tau Proteins/metabolism
13.
J Mol Biol ; 433(24): 167325, 2021 12 03.
Article in English | MEDLINE | ID: covidwho-1474745

ABSTRACT

Single domain proteins fold via diverse mechanisms emphasizing the intricate relationship between energetics and structure, which is a direct consequence of functional constraints and demands imposed at the level of sequence. On the other hand, elucidating the interplay between folding mechanisms and function is challenging in large proteins, given the inherent shortcomings in identifying metastable states experimentally and the sampling limitations associated with computational methods. Here, we show that free energy profiles and surfaces of large systems (>150 residues), as predicted by a statistical mechanical model, display a wide array of folding mechanisms with ubiquitous folding intermediates and heterogeneous native ensembles. Importantly, residues around the ligand binding or enzyme active site display a larger tendency to partially unfold and this manifests as intermediates or excited states along the folding coordinate in ligand binding domains, transcription repressors, and representative enzymes from all the six classes, including the SARS-CoV-2 receptor binding domain (RBD) of the spike protein and the protease Mpro. It thus appears that it is relatively easier to distill the imprints of function on the folding landscape of larger proteins as opposed to smaller systems. We discuss how an understanding of energetic-entropic features in ordered proteins can pinpoint specific avenues through which folding mechanisms, populations of partially structured states and function can be engineered.


Subject(s)
Enzymes/chemistry , Enzymes/metabolism , Models, Molecular , Protein Conformation , Protein Folding , Humans , Protein Binding , Protein Domains , Thermodynamics
14.
Cells ; 10(10)2021 10 03.
Article in English | MEDLINE | ID: covidwho-1444119

ABSTRACT

The data currently available on how the immune system recognises the SARS-CoV-2 virus is growing rapidly. While there are structures of some SARS-CoV-2 proteins in complex with antibodies, which helps us understand how the immune system is able to recognise this new virus; however, we lack data on how T cells are able to recognise this virus. T cells, especially the cytotoxic CD8+ T cells, are critical for viral recognition and clearance. Here we report the X-ray crystallography structure of a T cell receptor, shared among unrelated individuals (public TCR) in complex with a dominant spike-derived CD8+ T cell epitope (YLQ peptide). We show that YLQ activates a polyfunctional CD8+ T cell response in COVID-19 recovered patients. We detail the molecular basis for the shared TCR gene usage observed in HLA-A*02:01+ individuals, providing an understanding of TCR recognition towards a SARS-CoV-2 epitope. Interestingly, the YLQ peptide conformation did not change upon TCR binding, facilitating the high-affinity interaction observed.


Subject(s)
COVID-19/immunology , COVID-19/virology , Epitopes, T-Lymphocyte/chemistry , HLA-A2 Antigen/immunology , Receptors, Antigen, T-Cell/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , CD8-Positive T-Lymphocytes/cytology , Crystallography, X-Ray , Cytokines/metabolism , Epitopes/chemistry , HLA-A2 Antigen/chemistry , Humans , Mutation , Peptides/chemistry , Protein Binding , Protein Denaturation , Protein Folding , Surface Plasmon Resonance , T-Lymphocytes, Cytotoxic/immunology
16.
Nucleic Acids Res ; 49(W1): W425-W430, 2021 07 02.
Article in English | MEDLINE | ID: covidwho-1387966

ABSTRACT

Methods for estimating the quality of 3D models of proteins are vital tools for driving the acceptance and utility of predicted tertiary structures by the wider bioscience community. Here we describe the significant major updates to ModFOLD, which has maintained its position as a leading server for the prediction of global and local quality of 3D protein models, over the past decade (>20 000 unique external users). ModFOLD8 is the latest version of the server, which combines the strengths of multiple pure-single and quasi-single model methods. Improvements have been made to the web server interface and there has been successive increases in prediction accuracy, which were achieved through integration of newly developed scoring methods and advanced deep learning-based residue contact predictions. Each version of the ModFOLD server has been independently blind tested in the biennial CASP experiments, as well as being continuously evaluated via the CAMEO project. In CASP13 and CASP14, the ModFOLD7 and ModFOLD8 variants ranked among the top 10 quality estimation methods according to almost every official analysis. Prior to CASP14, ModFOLD8 was also applied for the evaluation of SARS-CoV-2 protein models as part of CASP Commons 2020 initiative. The ModFOLD8 server is freely available at: https://www.reading.ac.uk/bioinf/ModFOLD/.


Subject(s)
Computers , Models, Molecular , Neural Networks, Computer , Protein Conformation , Protein Folding , Proteins/chemistry , Software , Reproducibility of Results , Research Design , SARS-CoV-2/chemistry , Viral Proteins/chemistry
17.
Life Sci ; 281: 119774, 2021 Sep 15.
Article in English | MEDLINE | ID: covidwho-1284329

ABSTRACT

AIM: The present study attempts to decipher the site-specific amino acid alterations at certain positions experiencing preferential selectivity and their effect on proteins' stability and flexibility. The study examines the selection preferences by considering pair-wise non-bonded interaction energies of adjacent and interacting amino acids present at the interacting site, along with their evolutionary history. MATERIALS AND METHODS: For the study, variations in the interacting residues of spike protein (S-Protein) receptor-binding domain (RBD) of different coronaviruses were examined. The MD simulation trajectory analysis revealed that, though all the variants studied were structurally stable at their native and bound confirmations, the RBD of 2019-nCoV/SARS-CoV-2 was found to be more flexible and more dynamic. Furthermore, a noticeable change observed in the non-bonded interaction energies of the amino acids interacting with the receptor corroborated their selection at respective positions. KEY FINDINGS: The conformational changes exerted by the altered amino acids could be the reason for a broader range of interacting receptors among the selected proteins. SIGNIFICANCE: The results envisage a strong indication that the residue selection at certain positions is governed by a well-orchestrated feedback mechanism, which follows increased stability and flexibility in the folded structure compared to its evolutionary predecessor.


Subject(s)
Amino Acids/chemistry , Biological Evolution , Proteins/chemistry , Crystallography, X-Ray , Molecular Dynamics Simulation , Phylogeny , Protein Conformation , Protein Folding
18.
RNA ; 27(9): 1025-1045, 2021 09.
Article in English | MEDLINE | ID: covidwho-1269913

ABSTRACT

Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a nonstructural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation levels. We discover that a functionally coherent subset of human genes is preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5' terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5' UTRs from TOP transcripts can drive preferential expression in the presence of Nsp1. Finally, we found that LARP1, a key effector protein in the mTOR pathway, may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine-tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.


Subject(s)
Host-Pathogen Interactions/genetics , Protein Biosynthesis , Proteins/chemistry , Proteins/genetics , Viral Nonstructural Proteins/genetics , 5' Untranslated Regions , Autoantigens/genetics , Autoantigens/metabolism , Gene Expression Regulation , HEK293 Cells , Humans , Protein Folding , Pyrimidines , RNA, Messenger/genetics , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Ribosomes/genetics , Ribosomes/virology , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Viral Nonstructural Proteins/metabolism
19.
Nucleic Acids Res ; 49(W1): W589-W596, 2021 07 02.
Article in English | MEDLINE | ID: covidwho-1233865

ABSTRACT

ReFOLD3 is unique in its application of gradual restraints, calculated from local model quality estimates and contact predictions, which are used to guide the refinement of theoretical 3D protein models towards the native structures. ReFOLD3 achieves improved performance by using an iterative refinement protocol to fix incorrect residue contacts and local errors, including unusual bonds and angles, which are identified in the submitted models by our leading ModFOLD8 model quality assessment method. Following refinement, the likely resulting improvements to the submitted models are recognized by ModFOLD8, which produces both global and local quality estimates. During the CASP14 prediction season (May-Aug 2020), we used the ReFOLD3 protocol to refine hundreds of 3D models, for both the refinement and the main tertiary structure prediction categories. Our group improved the global and local quality scores for numerous starting models in the refinement category, where we ranked in the top 10 according to the official assessment. The ReFOLD3 protocol was also used for the refinement of the SARS-CoV-2 targets as a part of the CASP Commons COVID-19 initiative, and we provided a significant number of the top 10 models. The ReFOLD3 web server is freely available at https://www.reading.ac.uk/bioinf/ReFOLD/.


Subject(s)
Computers , Internet , Models, Molecular , Protein Conformation , Protein Folding , Proteins/chemistry , Software , Reproducibility of Results , SARS-CoV-2/chemistry , Viral Proteins/chemistry
20.
Science ; 372(6548): 1306-1313, 2021 06 18.
Article in English | MEDLINE | ID: covidwho-1228853

ABSTRACT

Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.


Subject(s)
Frameshifting, Ribosomal , RNA, Viral/genetics , Ribosomes/ultrastructure , SARS-CoV-2/genetics , Viral Proteins/biosynthesis , Animals , Antiviral Agents/pharmacology , Codon, Terminator , Coronavirus RNA-Dependent RNA Polymerase/biosynthesis , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Cryoelectron Microscopy , Fluoroquinolones/pharmacology , Frameshifting, Ribosomal/drug effects , Genome, Viral , Humans , Image Processing, Computer-Assisted , Models, Molecular , Nucleic Acid Conformation , Open Reading Frames , Protein Folding , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Ribosomal, 18S/chemistry , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 18S/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Viral Proteins/chemistry , Viral Proteins/genetics , Virus Replication/drug effects
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