Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Two adjacent mutations on the dimer interface of SARS coronavirus 3C-like protease cause different conformational changes in crystal structure.

ABSTRACT: The 3C-like protease of SARS coronavirus (SARS-CoV 3CL(pro)) is vital for SARS-CoV replication and is a promising drug target. It has been extensively proved that only the dimeric enzyme is active. Here we discovered that two adjacent mutations (Ser139_Ala and Phe140_Ala) on the dimer interface resulted in completely different crystal structures of the enzyme, demonstrating the distinct roles of these two residues in maintaining the active conformation of SARS-CoV 3CL(pro). S139A is a monomer that is structurally similar to the two reported monomers G11A and R298A. However, this mutant still retains a small fraction of dimer in solution, which might account for its remaining activity. F140A is a dimer with the most collapsed active pocket discovered so far, well-reflecting the stabilizing role of this residue. Moreover, a plausible dimerization mechanism was also deduced from structural analysis. Our work is expected to provide insight on the dimerization-function relationship of SARS-CoV 3CL(pro).

SUBMITTER: Hu T

PROVIDER: S-EPMC7103376 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Residues on the dimer interface of SARS coronavirus 3C-like protease: dimer stability characterization and enzyme catalytic activity analysis.

Project description:3C-like protease (3CL pro) plays pivotal roles in the life cycle of severe acute respiratory syndrome coronavirus (SARS-CoV) and only the dimeric protease is proposed as the functional form. Guided by the crystal structure and molecular dynamics simulations, we performed systematic mutation analyses to identify residues critical for 3CL pro dimerization and activity in this study. Seven residues on the dimer interface were selected for evaluating their contributions to dimer stability and catalytic activity by biophysical and biochemical methods. These residues are involved in dimerization through hydrogen bonding and broadly located in the N-terminal finger, the alpha-helix A' of domain I, and the oxyanion loop near the S1 substrate-binding subsite in domain II. We revealed that all seven single mutated proteases still have the dimeric species but the monomer-dimer equilibria of these mutants vary from each other, implying that these residues might contribute differently to the dimer stability. Such a conclusion could be further verified by the results that the proteolytic activities of these mutants also decrease to varying degrees. The present study would help us better understand the dimerization-activity relationship of SARS-CoV 3CL pro and afford potential information for designing anti-viral compounds targeting the dimer interface of the protease.

| S-EPMC7109808 | biostudies-literature

Mutation of Gly-11 on the dimer interface results in the complete crystallographic dimer dissociation of severe acute respiratory syndrome coronavirus 3C-like protease: crystal structure with molecular dynamics simulations.

Project description:SARS-CoV 3C-like protease (3CL(pro)) is an attractive target for anti-severe acute respiratory syndrome (SARS) drug discovery, and its dimerization has been extensively proved to be indispensable for enzymatic activity. However, the reason why the dissociated monomer is inactive still remains unclear due to the absence of the monomer structure. In this study, we showed that mutation of the dimer-interface residue Gly-11 to alanine entirely abolished the activity of SARS-CoV 3CL(pro). Subsequently, we determined the crystal structure of this mutant and discovered a complete crystallographic dimer dissociation of SARS-CoV 3CL(pro). The mutation might shorten the alpha-helix A' of domain I and cause a mis-oriented N-terminal finger that could not correctly squeeze into the pocket of another monomer during dimerization, thus destabilizing the dimer structure. Several structural features essential for catalysis and substrate recognition are severely impaired in the G11A monomer. Moreover, domain III rotates dramatically against the chymotrypsin fold compared with the dimer, from which we proposed a putative dimerization model for SARS-CoV 3CL(pro). As the first reported monomer structure for SARS-CoV 3CL(pro), the crystal structure of G11A mutant might provide insight into the dimerization mechanism of the protease and supply direct structural evidence for the incompetence of the dissociated monomer.

| S-EPMC7982321 | biostudies-literature

Crystal structure of human enterovirus 71 3C protease.

Project description:Human enterovirus 71 (EV71) is the major pathogen that causes hand, foot and mouth disease that particularly affects young children. Growing hand, foot and mouth disease outbreaks were observed worldwide in recent years and caused devastating losses both economically and politically. However, vaccines or effective drugs are unavailable to date. The genome of EV71 consists of a positive sense, single-stranded RNA of ∼7400 bp, encoding a large precursor polyprotein that requires proteolytic processing to generate mature viral proteins. The proteolytic processing mainly depends on EV71 3C protease (3C(pro)) that possesses both proteolysis and RNA binding activities, which enable the protease to perform multiple tasks in viral replication and pathogen-host interactions. The central roles played by EV71 3C(pro) make it an appealing target for antiviral drug development. We determined the first crystal structure of EV71 3C(pro) and analyzed its enzymatic activity. The crystal structure shows that EV71 3C(pro) has a typical chymotrypsin-like fold that is common in picornaviral 3C(pro). Strikingly, we found an important surface loop, also denoted as β-ribbon, which adopts a novel open conformation in EV71 3C(pro). We identified two important residues located at the base of the β-ribbon, Gly123 and His133, which form hinges that govern the intrinsic flexibility of the ribbon. Structure-guided mutagenesis studies revealed that the hinge residues are important to EV71 3C(pro) proteolytic activities. In summary, our work provides the first structural insight into EV71 3C(pro), including a mobile β-ribbon, which is relevant to the proteolytic mechanism. Our data also provides a framework for structure-guided inhibitor design against EV71 3C(pro).

| S-EPMC7094522 | biostudies-literature

HIV-1 protease dimer interface mutations that compensate for viral reverse transcriptase instability in infectious virions.

Project description:Mature enzymes encoded within the human immunodeficiency virus type 1 (HIV-1) genome (protease (PR), reverse transcriptase (RT) and integrase (IN)) derive from proteolytic processing of a large polyprotein (Gag-Pol). Gag-Pol processing is catalyzed by the viral PR, which is active as a homodimer. The HIV-1 RT functions as a heterodimer (p66/p51) composed of subunits of 560 and 440 amino acid residues, respectively. Both subunits have identical amino acid sequence, but p51 lacks 120 residues that are removed by the HIV-1 PR during viral maturation. While p66 is the catalytic subunit, p51 has a primarily structural role. Amino acid substitutions affecting the stability of p66/p51 (i.e. F130W) have a deleterious effect on viral fitness. Previously, we showed that the effects of F130W are mediated by p51 and can be compensated by mutation T58S. While studying the dynamics of emergence of the compensatory mutation, we observed that mutations in the viral PR-coding region were selected in HIV clones containing the RT substitution F130W, before the imposition of T58S/F130W mutations. The PR mutations identified (G94S and T96S) improved the replication capacity of the F130W mutant virus. By using a trans-complementation assay, we demonstrate that the loss of p66/p51 heterodimer stability caused by Trp130 can be attributed to an increased susceptibility of RT to viral PR degradation. Recombinant HIV-1 PRs bearing mutations G94S or T96S showed decreased dimer stability and reduced catalytic efficiency. These results were consistent with crystallographic data showing the location of both residues in the PR dimerization interface.

| S-EPMC2696282 | biostudies-literature

Mechanism for controlling the dimer-monomer switch and coupling dimerization to catalysis of the severe acute respiratory syndrome coronavirus 3C-like protease.

Project description:Unlike 3C protease, the severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CLpro) is only enzymatically active as a homodimer and its catalysis is under extensive regulation by the unique extra domain. Despite intense studies, two puzzles still remain: (i) how the dimer-monomer switch is controlled and (ii) why dimerization is absolutely required for catalysis. Here we report the monomeric crystal structure of the SARS-CoV 3CLpro mutant R298A at a resolution of 1.75 A. Detailed analysis reveals that Arg298 serves as a key component for maintaining dimerization, and consequently, its mutation will trigger a cooperative switch from a dimer to a monomer. The monomeric enzyme is irreversibly inactivated because its catalytic machinery is frozen in the collapsed state, characteristic of the formation of a short 3(10)-helix from an active-site loop. Remarkably, dimerization appears to be coupled to catalysis in 3CLpro through the use of overlapped residues for two networks, one for dimerization and another for the catalysis.

| S-EPMC2293028 | biostudies-literature

Crystal structure of SARS-CoV 3C-like protease with baicalein.

Project description:The 3C-like protease (Mpro, 3CLpro) plays a key role in the replication process in coronaviruses (CoVs). The Mpro is an essential enzyme mediates CoVs replication and is a promising target for development of antiviral drugs. Until now, baicalein has been shown the specific activity for SARS-CoV Mpro in vitro experiments. In this study, we resolved the SARS-CoV Mpro with baicalein by X-ray diffraction at 2.25 Å (PDB code 7XAX), which provided a structural basis for the research and development of baicalein as an anti-CoVs drug.

| S-EPMC9027212 | biostudies-literature

QSAR modeling and pharmacoinformatics of SARS coronavirus 3C-like protease inhibitors.

Project description:The search for effective treatment against novel coronavirus (COVID-19) remains a global challenge due to controversies on available vaccines. In this study, data of SARS coronavirus 3C-like protease (3CLpro) inhibitors; a key drug target in the coronavirus genome was retrieved from CHEMBL database. Quantitative Structure-Activity Relationship (QSAR) studies, Molecular docking, Absorption-Distribution-Metabolism-Excretion-Toxicity (ADMET) and molecular dynamics simulation (MDS) were carried out using these 3CLpro inhibitors. QSAR model constructed using the data had correlation coefficient R2 value of 0.907; cross-validated correlation coefficient Q2 value of 0.866 and test set predicted correlation coefficient R2pred value of 0.517. Variance inflation factor (VIF) values for descriptors contained in the model ranged from 1.352 to 1.68, hence, these descriptors were orthogonal to one another. Therefore, the model was statistically significant and can be used to screen and design new molecules for their inhibitory activity against 3CLpro. Molecular docking showed that seven of the compounds (inhibitors) used in the study had a remarkable binding affinity (-9.2 to -10.3 kcal/mol) for 3CLpro. ADMET study revealed that five (CHEMBL Accession IDs 19438, 196635, 377150, 208763, and 210097) of the seven compounds with good binding ability obeyed Lipinski's rule of five. Hence, they were compounds with drug-like properties. MDS analysis revealed that 3CLpro-compound 21, 3CLpro-compound 22, 3CLpro-compound 40 complexes are very stable as compared to the reference 3CLpro-X77 complex. Therefore, this study identified three potent inhibitors of 3CLpro viz. CHEMBL194398, CHEMBL196635, and CHEMBL210097 that can be further explored for the treatment of COVID-19.

| S-EPMC8111888 | biostudies-literature

Genetic screen for monitoring severe acute respiratory syndrome coronavirus 3C-like protease.

Project description:A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome. Site-specific proteolysis plays a critical role in regulating a number of cellular and viral processes. Since the main protease of SCoV, also termed 3C-like protease, is an attractive target for drug therapy, we have developed a safe, simple, and rapid genetic screen assay to monitor the activity of the SCoV 3C-like protease. This genetic system is based on the bacteriophage lambda regulatory circuit, in which the viral repressor cI is specifically cleaved to initiate the lysogenic-to-lytic switch. A specific target for the SCoV 3C-like protease, P1/P2 (SAVLQ/SGFRK), was inserted into the lambda phage cI repressor. The target specificity of the SCoV P1/P2 repressor was evaluated by coexpression of this repressor with a chemically synthesized SCoV 3C-like protease gene construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI. SCoV P1/P2-cro and the beta-galactosidase-SCoV 3C-like protease constructs, lambda phage replicated up to 2,000-fold more efficiently than in cells that did not express the SCoV 3C-like protease. This simple and highly specific assay can be used to monitor the activity of the SCoV 3C-like protease, and it has the potential to be used for screening specific inhibitors.

| S-EPMC533918 | biostudies-literature

Prediction of coronavirus 3C-like protease cleavage sites using machine-learning algorithms.

Project description:The coronavirus 3C-like (3CL) protease, a cysteine protease, plays an important role in viral infection and immune escape. However, there is still a lack of effective tools for determining the cleavage sites of the 3CL protease. This study systematically investigated the diversity of the cleavage sites of the coronavirus 3CL protease on the viral polyprotein, and found that the cleavage motif were highly conserved for viruses in the genera of Alphacoronavirus, Betacoronavirus and Gammacoronavirus. Strong residue preferences were observed at the neighboring positions of the cleavage sites. A random forest (RF) model was built to predict the cleavage sites of the coronavirus 3CL protease based on the representation of residues in cleavage motifs by amino acid indexes, and the model achieved an AUC of 0.96 in cross-validations. The RF model was further tested on an independent test dataset which were composed of cleavage sites on 99 proteins from multiple coronavirus hosts. It achieved an AUC of 0.95 and predicted correctly 80% of the cleavage sites. Then, 1,352 human proteins were predicted to be cleaved by the 3CL protease by the RF model. These proteins were enriched in several GO terms related to the cytoskeleton, such as the microtubule, actin and tubulin. Finally, a webserver named 3CLP was built to predict the cleavage sites of the coronavirus 3CL protease based on the RF model. Overall, the study provides an effective tool for identifying cleavage sites of the 3CL protease and provides insights into the molecular mechanism underlying the pathogenicity of coronaviruses.

| S-EPMC9060714 | biostudies-literature

Flavonoid-mediated inhibition of SARS coronavirus 3C-like protease expressed in Pichia pastoris.

Project description:The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Recombinant 3CL(pro) was expressed in Pichia pastoris GS115 as a 42 kDa protein that displayed a K ( m ) of 15 ± 2 ?M with Dabcyl-KTSAVLQSGFRKME-Edans as substrate. Purified 3CL(pro) was used for inhibition and kinetic assays with seven flavonoid compounds. The IC(50) of six flavonoid compounds were 47-381 ?M. Quercetin, epigallocatechin gallate and gallocatechin gallate (GCG) displayed good inhibition toward 3CL(pro) with IC(50) values of 73, 73 and 47 ?M, respectively. GCG showed a competitive inhibition pattern with K ( i ) value of 25 ± 1.7 ?M. In molecular docking experiments, GCG displayed a binding energy of -14 kcal mol(-1) to the active site of 3CL(pro) and the galloyl moiety at 3-OH position was required for 3CL(pro) inhibition activity.

| S-EPMC7087583 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data