scholarly journals Structural, energetic and lipophilic analysis of SARS-CoV-2 non-structural protein 9 (NSP9)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jéssica de O. Araújo ◽  
Silvana Pinheiro ◽  
William J. Zamora ◽  
Cláudio Nahum Alves ◽  
Jerônimo Lameira ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Critical Role ◽  
Drug Binding ◽  
Nucleic Acid Binding ◽  
Structural Protein ◽  
Hydrophobic Region ◽  
Hydrogen Bond Interactions ◽  
Treatment And Prevention ◽  
Dynamics Simulations

AbstractIn SARS-CoV-2 replication complex, the Non-structural protein 9 (Nsp9) is an important RNA binding subunit in the RNA-synthesizing machinery. The dimeric forms of coronavirus Nsp9 increase their nucleic acid binding affinity and the N-finger motif appears to play a critical role in dimerization. Here, we present a structural, lipophilic and energetic study about the Nsp9 dimer of SARS-CoV-2 through computational methods that complement hydrophobicity scales of amino acids with molecular dynamics simulations. Additionally, we presented a virtual N-finger mutation to investigate whether this motif contributes to dimer stability. The results reveal for the native dimer that the N-finger contributes favorably through hydrogen bond interactions and two amino acids bellowing to the hydrophobic region, Leu45 and Leu106, are crucial in the formation of the cavity for potential drug binding. On the other hand, Gly100 and Gly104, are responsible for stabilizing the α-helices and making the dimer interface remain stable in both, native and mutant (without N-finger motif) systems. Besides, clustering results for the native dimer showed accessible cavities to drugs. In addition, the energetic and lipophilic analysis reveal that the higher binding energy in the native dimer can be deduced since it is more lipophilic than the mutant one, increasing non-polar interactions, which is in line with the result of MM-GBSA and SIE approaches where the van der Waals energy term has the greatest weight in the stability of the native dimer. Overall, we provide a detailed study on the Nsp9 dimer of SARS-CoV-2 that may aid in the development of new strategies for the treatment and prevention of COVID-19.

Clustering and Sampling of the c-Met Conformational Space: A Computational Drug Discovery Study

2020 ◽  
Vol 22 (9) ◽  
pp. 635-648 ◽  
Author(s):  
Korosh Mashayekh ◽  
Shahrzad Sharifi ◽  
Tahereh Damghani ◽  
Maryam Elyasi ◽  
Mohammad S. Avestan ◽  
...  
Keyword(s):  
Critical Role ◽  
Scientific Work ◽  
Binding Mode ◽  
Docking Studies ◽  
Conformational Space ◽  
Hydrogen Bond Interactions ◽  
Docking Program ◽  
Dynamics Simulations ◽  
Computational Drug Discovery ◽  
Dynamic Ensembles

Background: c-Met kinase plays a critical role in a myriad of human cancers, and a massive scientific work was devoted to design more potent inhibitors. Objective: In this study, 16 molecular dynamics simulations of different complexes of potent c-Met inhibitors with U-shaped binding mode were carried out regarding the dynamic ensembles to design novel potent inhibitors. Methods: A cluster analysis was performed, and the most representative frame of each complex was subjected to the structure-based pharmacophore screening. The GOLD docking program investigated the interaction energy and pattern of output hits from the virtual screening. The most promising hits with the highest scoring values that showed critical interactions with c-Met were presented for ADME/Tox analysis. Results: The screening yielded 45,324 hits that all of them were subjected to the docking studies and 10 of them with the highest-scoring values having diverse structures were presented for ADME/Tox analyses. Conclusion: The results indicated that all the hits shared critical Pi-Pi stacked and hydrogen bond interactions with Tyr1230 and Met1160 respectively.

scholarly journals Crystal Structure of Nonstructural Protein 10 from the Severe Acute Respiratory Syndrome Coronavirus Reveals a Novel Fold with Two Zinc-Binding Motifs

2006 ◽  
Vol 80 (16) ◽  
pp. 7894-7901 ◽  
Author(s):  
Jeremiah S. Joseph ◽  
Kumar Singh Saikatendu ◽  
Vanitha Subramanian ◽  
Benjamin W. Neuman ◽  
Alexei Brooun ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Severe Acute Respiratory Syndrome ◽  
Structural Integrity ◽  
Rna Binding ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Critical Role ◽  
Sequence Specificity ◽  
Nucleic Acid Binding ◽  
Murine Hepatitis Virus

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) possesses a large 29.7-kb positive-stranded RNA genome. The first open reading frame encodes replicase polyproteins 1a and 1ab, which are cleaved to generate 16 “nonstructural” proteins, nsp1 to nsp16, involved in viral replication and/or RNA processing. Among these, nsp10 plays a critical role in minus-strand RNA synthesis in a related coronavirus, murine hepatitis virus. Here, we report the crystal structure of SARS-CoV nsp10 at a resolution of 1.8 Å as determined by single-wavelength anomalous dispersion using phases derived from hexatantalum dodecabromide. nsp10 is a single domain protein consisting of a pair of antiparallel N-terminal helices stacked against an irregular β-sheet, a coil-rich C terminus, and two Zn fingers. nsp10 represents a novel fold and is the first structural representative of this family of Zn finger proteins found so far exclusively in coronaviruses. The first Zn finger coordinates a Zn2+ ion in a unique conformation. The second Zn finger, with four cysteines, is a distant member of the “gag-knuckle fold group” of Zn2+-binding domains and appears to maintain the structural integrity of the C-terminal tail. A distinct clustering of basic residues on the protein surface suggests a nucleic acid-binding function. Gel shift assays indicate that in isolation, nsp10 binds single- and double-stranded RNA and DNA with high-micromolar affinity and without obvious sequence specificity. It is possible that nsp10 functions within a larger RNA-binding protein complex. However, its exact role within the replicase complex is still not clear.

The in Silicon Cloning ILF2 Gene and Prediction of ILF2 Protein Structure in Sheep

2011 ◽  
Vol 345 ◽  
pp. 423-428
Author(s):  
Ying Ning Sun ◽  
Yu Zhao ◽  
Wei Yu Wang
Keyword(s):  
Amino Acids ◽  
Gene Sequence ◽  
Rna Binding ◽  
Binding Domain ◽  
Nucleic Acid Binding ◽  
Zebra Fish ◽  
Probe Sequence ◽  
Base Pairs ◽  
Reading Frame ◽  
Cloned Cdna

In silicon cloning, we obtained ILF2 gene by using human ILF2 gene sequence (NM_004515) to be probe. Sequence analysis showed that the in silicon cloned cDNA was 1662 base pairs long with an open reading frame (ORF) containing 1173 nucleotides encoding a protein of 390 amino acids. 5’-untranslated region (UTR) was 74 bp, and 3’-UTR was 413 bp. A comparison of the sheep ILF2 with cow, horse, human, mouse, xenopus and zebra fish ILF2 amino acids had 96%, 91%, 91%, 81%, 61%, and 54% identity. The PI was 5.19, and molecular weight of the deduced protein was 43 050.12 Da. The pig ILF2 contained a RGG-rich single-stranded RNA-binding domain and a DZF zinc-finger nucleic acid binding domain. This study laid a foundation for further analysis of structure, expression and regulation of ILF2 gene in sheep.

scholarly journals Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN

2021 ◽  
Author(s):  
Nicholas H Moeller ◽  
Ke Shi ◽  
Özlem Demir ◽  
Surajit Banerjee ◽  
Lulu Yin ◽  
...  
Keyword(s):  
Active Site ◽  
Rna Binding ◽  
Viral Rna ◽  
Rna Recombination ◽  
Structural Protein ◽  
Template Strand ◽  
Viral Virulence ◽  
High Resolution Structure ◽  
Dynamics Simulations ◽  
Computational Analyses

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in non-structural protein 14 (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-Å resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.

scholarly journals Targeting of Non-Structural Protein 9 as a Novel Therapeutic Target for the Treatment of SARS-CoV-2

2020 ◽  
Vol 3 ◽  
Author(s):  
Matthew Anderson ◽  
John Turchi
Keyword(s):  
Viral Replication ◽  
Therapeutic Target ◽  
Rna Binding ◽  
Future Research ◽  
Great Promise ◽  
Dependent Manner ◽  
Nucleic Acid Binding ◽  
Structural Protein ◽  
Concentration Dependent Manner ◽  
Novel Therapeutic

Background/Hypothesis:  The 2019 novel coronavirus (SARS-CoV-2) is a human coronavirus responsible for a global pandemic with over 13 million confirmed cases. Currently, there are no treatments to block viral infection or replication. Exploring novel therapeutic targets for SARS-CoV-2 and future coronaviruses holds great promise for treating the current and future outbreaks. One such target is the non-structural protein 9 (nsp9), which has been shown to be highly conserved and unique to the coronavirus family as well as playing a role in viral replication. We hypothesize nsp9 is a viable target for therapeutic development.     Methods:  Towards determining the utility of targeting nsp9, a series of databases were queried for articles pertaining to nsp9 in SARS-CoV-2 and other coronaviruses and coronaviruses in general. We assessed structural, biochemical and cellular features of nsp9.      Results:  Nsp9 forms a homodimer via a conserved a-helix containing a glycine-rich interaction motif (GxxxG). Dimerization at the GxxxG interface is required for efficient viral replication. Nsp9’s core is an open, six-stranded b-barrel whose fold gives it a structure similar to nucleic acid binding OB-fold proteins. This OB-like fold has not been detected in replicative complexes of other RNA viruses and may reflect the unique and complex CoV replication machinery. Nsp9 is an indispensable component of the replication complex that binds single-stranded RNA in a concentration-dependent manner. A recent bioinformatic approach also found that nsp9 interacts with NF-kappa-B-repressing factor and may play a role in the IL-8/IL-6 mediated chemotaxis of neutrophils and inflammatory response observed in Covid-19 patients.     Conclusion/Potential Impact:   Based on this research, we conclude nsp9 represents a novel therapeutic target whose OB-like-fold may provide a targetable structure for interrupting RNA binding and impairing viral replication. This study will help inform current and future research that seeks to target nsp9’s structure and biochemical interactions as treatment for coronavirus infection. 

scholarly journals Molecular Dynamics Simulations of Influenza A Virus NS1 Reveal a Remarkably Stable RNA-Binding Domain Harboring Promising Druggable Pockets

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 537
Author(s):  
Hiba Abi Hussein ◽  
Colette Geneix ◽  
Camille Cauvin ◽  
Daniel Marc ◽  
Delphine Flatters ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Influenza A ◽  
Rna Binding ◽  
Biological Activities ◽  
Binding Domain ◽  
Structural Protein ◽  
Binding Interface ◽  
Rna Binding Domain ◽  
Dynamics Simulations

The non-structural protein NS1 of influenza A viruses is considered to be the major antagonist of the interferon system and antiviral defenses of the cell. It could therefore represent a suitable target for novel antiviral strategies. As a first step towards the identification of small compounds targeting NS1, we here investigated the druggable potential of its RNA-binding domain since this domain is essential to the biological activities of NS1. We explored the flexibility of the full-length protein by running molecular dynamics simulations on one of its published crystal structures. While the RNA-binding domain structure was remarkably stable along the simulations, we identified a flexible site at the two extremities of the “groove” that is delimited by the antiparallel α-helices that make up its RNA-binding interface. This groove region is able to form potential binding pockets, which, in 60% of the conformations, meet the druggability criteria. We characterized these pockets and identified the residues that contribute to their druggability. All the residues involved in the druggable pockets are essential at the same time to the stability of the RNA-binding domain and to the biological activities of NS1. They are also strictly conserved across the large sequence diversity of NS1, emphasizing the robustness of this search towards the identification of broadly active NS1-targeting compounds.

scholarly journals Mutagenesis of hepatitis C virus E1 protein affects its membrane-permeabilizing activity

2001 ◽  
Vol 82 (9) ◽  
pp. 2243-2250 ◽  
Author(s):  
A. R. Ciccaglione ◽  
A. Costantino ◽  
C. Marcantonio ◽  
M. Equestre ◽  
A. Geraci ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Membrane Permeability ◽  
Protein Function ◽  
Critical Role ◽  
Cell Lysis ◽  
Hydrophobic Region ◽  
E Coli ◽  
Conserved Amino Acids

The E1 glycoprotein of hepatitis C virus is a transmembrane glycoprotein with a C-terminal anchor domain. When expressed in Escherichia coli, E1 induces a change in membrane permeability that is toxic to the bacterial cell. The C-terminal hydrophobic region (aa 331–383) of E1 is mainly responsible for membrane association and for inducing changes in membrane permeability. These observed changes are similar to those produced in E. coli by influenza virus M2, human immunodeficiency virus gp41 and poliovirus 3AB proteins, whose hydrophobic domains are thought to cause pore formation in biological membranes. To further characterize the activity of E1 at a molecular level, the membrane-permeabilizing ability of a second internal hydrophobic region (aa 262–291) was examined by expressing different deletion mutants of E1 in an E. coli system that is widely used for analysing membrane-active proteins from other animal viruses. Moreover, highly conserved amino acids in the C-terminal hydrophobic region were mutated to identify residues that are critical for inducing changes in membrane permeability. Analysis of cell growth curves of recombinant cultures and membrane-permeability assays revealed that synthesis of this fragment increased the flux of small compounds through the membrane and caused progressive cell lysis, suggesting that this domain has membrane-active properties. Furthermore, analysis of C-terminal mutants indicated that the conserved amino acids Arg339, Trp368 and Lys370 play a critical role in protein function, as both cell lysis and changes in membrane permeability induced by the wild-type clone could be blocked by substitutions in these positions.

scholarly journals Dimerization of Coronavirus nsp9 with Diverse Modes Enhances Its Nucleic Acid Binding Affinity

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Zhe Zeng ◽  
Feng Deng ◽  
Ke Shi ◽  
Gang Ye ◽  
Gang Wang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Crystal Structures ◽  
Binding Affinity ◽  
Disulfide Bond ◽  
Rna Binding ◽  
Critical Role ◽  
Nucleic Acid Binding ◽  
Wild Type ◽  
Dimer Interface ◽  
Diarrhea Virus

ABSTRACTCoronaviruses pose serious health threats to humans and other animals. Understanding the mechanisms of their replication has important implications for global health and economic stability. Nonstructural protein 9 (nsp9) is an essential RNA binding protein for coronavirus replication. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. Here, we report four crystal structures, including wild-type porcine delta coronavirus (PDCoV) nsp9, PDCoV nsp9-ΔN7 (N-terminal 7 amino acids deleted), wild-type porcine epidemic diarrhea virus (PEDV) nsp9, and PEDV nsp9-C59A mutant. These structures reveal the diverse dimerization forms of coronavirus nsp9. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. Interestingly, size exclusion chromatography and analytical ultracentrifugation analyses indicate that the PDCoV nsp9-ΔN7 and PEDV nsp9-C59A mutants are monomeric in solution. In addition, electrophoretic mobility shift assays and microscale thermophoresis analysis indicate that the monomeric forms of PDCoV nsp9 and PEDV nsp9 still have nucleic acid binding affinity, although it is lower than that of the wild type. Our results show that the diverse dimerization forms of coronavirus nsp9 proteins enhance their nucleic acid binding affinity.IMPORTANCECoronaviruses cause widespread respiratory, gastrointestinal, and central nervous system diseases in humans and other animals, threatening human health and causing economic loss. Coronavirus nsp9, a member of the replication complex, is an important RNA binding subunit in the RNA-synthesizing machinery of all coronaviruses. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. In this study we determined the nsp9 crystal structures of PDCoV and PEDV. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. This study provides a structural and functional basis for understanding the mechanism of dimerization and shows that the diverse dimerization modes of coronavirus nsp9 proteins enhance their nucleic acid binding affinity. Importantly, these findings may provide a new insight for antiviral drug development.

scholarly journals Characterization of the nucleic acid-binding activity of the avian reovirus non-structural protein σNS

2005 ◽  
Vol 86 (4) ◽  
pp. 1159-1169 ◽  
Author(s):  
Fernando Tourís-Otero ◽  
José Martínez-Costas ◽  
Vikram N. Vakharia ◽  
Javier Benavente
Keyword(s):  
Insect Cells ◽  
Rna Binding ◽  
Binding Activity ◽  
Sequence Specificity ◽  
Avian Reovirus ◽  
Nucleic Acid Binding ◽  
Structural Protein ◽  
Independent Manner ◽  
Ribonucleoprotein Complexes

The avian reovirus non-structural protein σNS has previously been shown to bind single-stranded (ss) RNA in vitro in a sequence-independent manner. The results of the present study further reveal that σNS binds poly(A), poly(U) and ssDNA, but not poly(C), poly(G) or duplex nucleic acids, suggesting that σNS has some nucleotide-sequence specificity for ssRNA binding. The current findings also show that σNS is present in large ribonucleoprotein complexes in the cytoplasm of avian reovirus-infected cells, indicating that it exists in intimate association with ssRNAs in vivo. Removal of RNA from the complexes generates a σNS protein form that sediments between 4·5 and 7 S, suggesting that RNA-free σNS associates into small oligomers. Expression and purification of recombinant σNS in insect cells allowed us to generate specific antibodies and to perform a variety of assays. The results of these assays revealed that: (i) RNA-free σNS exists as homodimers and homotrimers; (ii) the minimum RNA size for σNS binding is between 10 and 20 nt; (iii) σNS does not have a preference for viral mRNA sequences; and (iv) its RNA-binding activity is conformation-dependent. Baculovirus expression of point and deletion σNS mutants in insect cells showed that the five conserved basic amino acids that are important for RNA binding and ribonucleoprotein-complex formation are dispersed throughout the entire σNS sequence, suggesting that this protein binds ssRNA through conformational domains. Finally, the properties of the avian reovirus protein σNS are compared with those of its mammalian reovirus counterpart.

Use of Molecular Dynamics Simulations in Structure-Based Drug Discovery

2019 ◽  
Vol 25 (31) ◽  
pp. 3339-3349 ◽  
Author(s):  
Indrani Bera ◽  
Pavan V. Payghan
Keyword(s):  
Molecular Dynamics ◽  
Drug Discovery ◽  
Molecular Dynamics Simulations ◽  
Drug Target ◽  
Structural Information ◽  
Drug Binding ◽  
Structure Based Drug Design ◽  
Drug Designing ◽  
Target Binding ◽  
Dynamics Simulations

Background: Traditional drug discovery is a lengthy process which involves a huge amount of resources. Modern-day drug discovers various multidisciplinary approaches amongst which, computational ligand and structure-based drug designing methods contribute significantly. Structure-based drug designing techniques require the knowledge of structural information of drug target and drug-target complexes. Proper understanding of drug-target binding requires the flexibility of both ligand and receptor to be incorporated. Molecular docking refers to the static picture of the drug-target complex(es). Molecular dynamics, on the other hand, introduces flexibility to understand the drug binding process. Objective: The aim of the present study is to provide a systematic review on the usage of molecular dynamics simulations to aid the process of structure-based drug design. Method: This review discussed findings from various research articles and review papers on the use of molecular dynamics in drug discovery. All efforts highlight the practical grounds for which molecular dynamics simulations are used in drug designing program. In summary, various aspects of the use of molecular dynamics simulations that underline the basis of studying drug-target complexes were thoroughly explained. Results: This review is the result of reviewing more than a hundred papers. It summarizes various problems that use molecular dynamics simulations. Conclusion: The findings of this review highlight how molecular dynamics simulations have been successfully implemented to study the structure-function details of specific drug-target complexes. It also identifies the key areas such as stability of drug-target complexes, ligand binding kinetics and identification of allosteric sites which have been elucidated using molecular dynamics simulations.

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