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 Computational Guided Identification of Novel Potent Inhibitors of NTD-N-Protein of SARS-CoV-2

2020 ◽  
Author(s):  
Poonam Dhankhar ◽  
vikram dalal ◽  
Vishakha Singh ◽  
Shailly Tomar ◽  
pravindra kumar
Keyword(s):  
Rna Binding ◽  
Pharmacophore Model ◽  
Guanosine Monophosphate ◽  
Viral Rna ◽  
The Novel ◽  
N Protein ◽  
Legitimate Target ◽  
Zinc Database ◽  
Dynamics Simulations ◽  
Virus Replication Cycle

<p>The Coronavirus Disease 2019 (COVID-19), caused by the SARS-CoV-2 virus has raised severe health problems in china and across the world as well. CoVs encode the nucleocapsid protein (N-protein), an essential RNA-binding protein that performs different roles throughout the virus replication cycle and forms the ribonucleoprotein complex with viral RNA using the N-terminal domain (NTD) of N-protein. Recent studies have shown that NTD-N-protein is a legitimate target for the development of antiviral drugs against human CoVs. Owing to the importance of NTD, the present study focuses on targeting the NTD-N-protein from SARS-CoV-2 to identify the potential compounds. The pharmacophore model has been developed based on the guanosine monophosphate (GMP), a RNA substrate and further pharmacophore-based virtual screening was performed against ZINC database. The screened compounds were filtered by analysing the <i>in silico</i> ADMET properties and drug-like properties. The pharmacokinetically screened compounds (ZINC000257324845, ZINC000005169973, and ZINC000009913056) were further scrutinized through computational approaches including molecular docking and molecular dynamics simulations and revealed that these compounds exhibited good binding affinity as compared to GMP and provide stability to their respective complex with the NTD. Our findings could disrupt the binding of viral RNA to NTD, which may inhibit the essential functions of NTD. These findings may further provide an impetus to develop the novel and potential inhibitor against SARS-CoV-2.<br></p>

scholarly journals Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus genome segment 9

2002 ◽  
Vol 83 (6) ◽  
pp. 1483-1491 ◽  
Author(s):  
Kaustubha R. Qanungo ◽  
Subhas C. Kundu ◽  
James I. Mullins ◽  
Ananta K. Ghosh
Keyword(s):  
Structure Prediction ◽  
Rna Binding ◽  
Secondary Structure Prediction ◽  
Virus Genome ◽  
Genome Segment ◽  
Viral Rna ◽  
Structural Protein ◽  
Antheraea Mylitta ◽  
Cytoplasmic Polyhedrosis Virus ◽  
Polyhedrosis Virus

Genome segment 9 of the 11-segment RNA genomes of three cytoplasmic polyhedrosis virus (CPV) isolates from Antheraea mylitta (AmCPV), Antheraea assamensis (AaCPV) and Antheraea proylei (ApCPV) were converted to cDNA, cloned and sequenced. In each case, this genome segment consists of 1473 nucleotides with one long ORF of 1035 bp and encodes a protein of 345 amino acids, termed NSP38, with a molecular mass of 38 kDa. Secondary structure prediction showed the presence of nine α-helices in the central and terminal domains with localized similarity to RNA-binding motifs of bluetongue virus and infectious bursal disease virus RNA polymerases. Nucleotide sequences were 99·6% identical between these three strains of CPVs, but no similarity was found to any other nucleotide or protein sequence in public databases. The ORF from AmCPV cDNA was expressed as a His-tagged fusion protein in E. coli and polyclonal antibody was raised against the purified protein. Immunoblot as well as immunofluorescence analysis with anti-NSP38 antibody showed that the protein was not present in polyhedra or uninfected cells but was present in AmCPV-infected host midgut cells. NSP38 was expressed in insect cells as soluble protein via a baculovirus expression vector and shown to possess the ability to bind poly(rI)–(rC) agarose, which was competitively removed by AmCPV viral RNA. These results indicate that NSP38 is expressed in virus-infected cells as a non-structural protein. By binding to viral RNA, it may play a role in the regulation of genomic RNA function and packaging.

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 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.

scholarly journals Two mutations P/L and Y/C in SARS-CoV-2 helicase domain exist together and influence helicase RNA binding

2020 ◽  
Author(s):  
Feroza Begum ◽  
Arup Kumar Banerjee ◽  
Prem Prakash Tripathi ◽  
Upasana Ray
Keyword(s):  
Rna Binding ◽  
Rna Virus ◽  
Novel Mutation ◽  
Rna Replication ◽  
Viral Rna ◽  
Helicase Domain ◽  
Dependent Manner ◽  
Structural Protein ◽  
Rna Helicases ◽  
Rna Interaction

AbstractRNA helicases play pivotal role in RNA replication by catalysing the unwinding of complex RNA duplex structures into single strands in ATP/NTP dependent manner. SARS coronavirus 2 (SARS-CoV-2) is a single stranded positive sense RNA virus belonging to the family Coronaviridae. The viral RNA encodes non structural protein Nsp13 or the viral helicase protein that helps the viral RNA dependent RNA polymerase (RdRp) to execute RNA replication by unwinding the RNA duplexes. In this study we identified a novel mutation at position 541of the helicase where the tyrosine (Y) got substituted with cytosine (C). We found that Y541C is a destabilizing mutation increasing the molecular flexibility and leading to decreased affinity of helicase binding with RNA. Earlier we had reported a mutation P504L in the helicase protein for which had not performed RNA binding study. Here we report that P504L mutation leads to increased affinity of helicase RNA interaction. So, both these mutations have opposite effects on RNA binding. Moreover, we found a significant fraction of isolate population where both P504L and Y541C mutations were co-existing.

scholarly journals Computational Guided Identification of Novel Potent Inhibitors of NTD-N-Protein of SARS-CoV-2

2020 ◽  
Author(s):  
Poonam Dhankhar ◽  
vikram dalal ◽  
Vishakha Singh ◽  
Shailly Tomar ◽  
pravindra kumar
Keyword(s):  
Rna Binding ◽  
Pharmacophore Model ◽  
Guanosine Monophosphate ◽  
Viral Rna ◽  
The Novel ◽  
N Protein ◽  
Legitimate Target ◽  
Zinc Database ◽  
Dynamics Simulations ◽  
Virus Replication Cycle

<p>The Coronavirus Disease 2019 (COVID-19), caused by the SARS-CoV-2 virus has raised severe health problems in china and across the world as well. CoVs encode the nucleocapsid protein (N-protein), an essential RNA-binding protein that performs different roles throughout the virus replication cycle and forms the ribonucleoprotein complex with viral RNA using the N-terminal domain (NTD) of N-protein. Recent studies have shown that NTD-N-protein is a legitimate target for the development of antiviral drugs against human CoVs. Owing to the importance of NTD, the present study focuses on targeting the NTD-N-protein from SARS-CoV-2 to identify the potential compounds. The pharmacophore model has been developed based on the guanosine monophosphate (GMP), a RNA substrate and further pharmacophore-based virtual screening was performed against ZINC database. The screened compounds were filtered by analysing the <i>in silico</i> ADMET properties and drug-like properties. The pharmacokinetically screened compounds (ZINC000257324845, ZINC000005169973, and ZINC000009913056) were further scrutinized through computational approaches including molecular docking and molecular dynamics simulations and revealed that these compounds exhibited good binding affinity as compared to GMP and provide stability to their respective complex with the NTD. Our findings could disrupt the binding of viral RNA to NTD, which may inhibit the essential functions of NTD. These findings may further provide an impetus to develop the novel and potential inhibitor against SARS-CoV-2.<br></p>

scholarly journals Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity

2021 ◽  
Vol 22 (9) ◽  
pp. 4769
Author(s):  
Pablo Maturana ◽  
María S. Orellana ◽  
Sixto M. Herrera ◽  
Ignacio Martínez ◽  
Maximiliano Figueroa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Conformational Dynamics ◽  
High Specificity ◽  
Arginine Decarboxylase ◽  
Space Groups ◽  
X Ray Crystallography ◽  
High Dynamics ◽  
Dynamics Simulations

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis

2019 ◽  
Vol 476 (6) ◽  
pp. 991-1003 ◽  
Author(s):  
Vijaykumar Pillalamarri ◽  
Tarun Arya ◽  
Neshatul Haque ◽  
Sandeep Chowdary Bala ◽  
Anil Kumar Marapaka ◽  
...  
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Covalent Modification ◽  
Structural Basis ◽  
Wild Type ◽  
Methionine Aminopeptidases ◽  
Synthetic Derivative ◽  
Dynamics Simulations

Abstract Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

WHICH IS THE PROTON-SHUTTLE IN ISOXANTHOPTERIN DEAMINASE? QM/MM MD UNDERSTANDING

2013 ◽  
Vol 12 (08) ◽  
pp. 1341002 ◽  
Author(s):  
XIN ZHANG ◽  
MING LEI
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Hydrogen Bonds ◽  
Proton Transfer ◽  
Glutamic Acid ◽  
Active Site ◽  
Nucleophilic Attack ◽  
Zinc Ions ◽  
Initial Model ◽  
Dynamics Simulations

The deamination process of isoxanthopterin catalyzed by isoxanthopterin deaminase was determined using the combined QM(PM3)/MM molecular dynamics simulations. In this paper, the updated PM3 parameters were employed for zinc ions and the initial model was built up based on the crystal structure. Proton transfer and following steps have been investigated in two paths: Asp336 and His285 serve as the proton shuttle, respectively. Our simulations showed that His285 is more effective than Aap336 in proton transfer for deamination of isoxanthopterin. As hydrogen bonds between the substrate and surrounding residues play a key role in nucleophilic attack, we suggested mutating Thr195 to glutamic acid, which could enhance the hydrogen bonds and help isoxanthopterin get close to the active site. The simulations which change the substrate to pterin 6-carboxylate also performed for comparison. Our results provide reference for understanding of the mechanism of deaminase and for enhancing the deamination rate of isoxanthopterin deaminase.

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