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- PDB-3eyd: Structure of HCV NS3-4A Protease with an Inhibitor Derived from a... -

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Basic information

Entry
Database: PDB / ID: 3eyd
TitleStructure of HCV NS3-4A Protease with an Inhibitor Derived from a Boronic Acid
Components
  • HCV NS3
  • HCV NS4a peptide
KeywordsVIRAL PROTEIN / Hepatitis C Virus / NS3 Protease Domain / serine protease / boronic acid inhibitor / Envelope protein / Helicase / Hydrolase / Nucleotide-binding / RNA replication / Transmembrane
Function / homology
Function and homology information


hepacivirin / host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated suppression of host TRAF-mediated signal transduction / transformation of host cell by virus / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / lipid droplet / ribonucleoside triphosphate phosphatase activity ...hepacivirin / host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated suppression of host TRAF-mediated signal transduction / transformation of host cell by virus / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / lipid droplet / ribonucleoside triphosphate phosphatase activity / SH3 domain binding / nucleoside-triphosphate phosphatase / channel activity / monoatomic ion transmembrane transport / viral nucleocapsid / clathrin-dependent endocytosis of virus by host cell / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / RNA helicase activity / host cell perinuclear region of cytoplasm / host cell endoplasmic reticulum membrane / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / induction by virus of host autophagy / ribonucleoprotein complex / RNA-directed RNA polymerase / viral translational frameshifting / viral RNA genome replication / cysteine-type endopeptidase activity / serine-type endopeptidase activity / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / fusion of virus membrane with host endosome membrane / viral envelope / host cell nucleus / virion attachment to host cell / apoptotic process / host cell plasma membrane / structural molecule activity / virion membrane / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
Thrombin, subunit H - #120 / Hepatitus C virus, Non-structural 5a protein, C-terminal / Hepatitis C virus NS5A, 1B domain superfamily / Hepatitis C virus non-structural protein NS2, N-terminal domain / Hepatitis C virus non-structural protein NS2 / HCV NS5a protein C-terminal region / Hepatitis C virus, Non-structural protein NS4b / Hepatitis C virus, Core protein, N-terminal / Hepatitis C virus non-structural protein NS2, C-terminal domain / Hepatitis C virus core protein, chain A superfamily ...Thrombin, subunit H - #120 / Hepatitus C virus, Non-structural 5a protein, C-terminal / Hepatitis C virus NS5A, 1B domain superfamily / Hepatitis C virus non-structural protein NS2, N-terminal domain / Hepatitis C virus non-structural protein NS2 / HCV NS5a protein C-terminal region / Hepatitis C virus, Non-structural protein NS4b / Hepatitis C virus, Core protein, N-terminal / Hepatitis C virus non-structural protein NS2, C-terminal domain / Hepatitis C virus core protein, chain A superfamily / : / Hepatitis C virus non-structural protein NS4b / Hepatitis C virus capsid protein / Hepatitis C virus, Non-structural 5a protein / Hepatitis C virus, Non-structural 5a protein, domain 1a / Hepatitis C virus non-structural 5a, 1B domain / NS5A domain 1a superfamily / Hepatitis C virus non-structural 5a zinc finger domain / Hepatitis C virus non-structural 5a domain 1b / Hepatitis C virus, Non-structural protein NS2 / : / NS3 RNA helicase, C-terminal helical domain / Hepacivirus nonstructural protein 2 (NS2) protease domain profile. / Hepatitis C virus non-structural 5a protein membrane anchor / Hepatitis C virus, Non-structural protein NS4a / Hepatitis C virus non-structural protein NS4a / Hepatitis C virus, Core protein, C-terminal / Hepatitis C virus core protein / Hepatitis C virus, Non-structural protein E2/NS1 / Hepatitis C virus non-structural protein E2/NS1 / Hepatitis C virus, Envelope glycoprotein E1 / Hepatitis C virus envelope glycoprotein E1 / RNA dependent RNA polymerase, hepatitis C virus / Viral RNA dependent RNA polymerase / Hepatitis C virus, NS3 protease, Peptidase S29 / Hepacivirus/Pegivirus NS3 protease domain profile. / Hepatitis C virus NS3 protease / DEAD box, Flavivirus / Flavivirus DEAD domain / helicase superfamily c-terminal domain / Trypsin-like serine proteases / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Thrombin, subunit H / Reverse transcriptase/Diguanylate cyclase domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / Beta Barrel / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta
Similarity search - Domain/homology
Chem-BE8 / Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesHepatitis C virus subtype 1a
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 2.3 Å
AuthorsVenkatraman, S. / Wu, W. / Prongay, A.J. / Girijavallabhan, V. / Njoroge, F.G.
CitationJournal: Bioorg.Med.Chem.Lett. / Year: 2009
Title: Potent inhibitors of HCV-NS3 protease derived from boronic acids.
Authors: Venkatraman, S. / Wu, W. / Prongay, A. / Girijavallabhan, V. / George Njoroge, F.
History
DepositionOct 20, 2008Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 10, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 20, 2021Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / struct_conn / struct_conn_type / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn_type.id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.3Dec 27, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Revision 1.4Apr 3, 2024Group: Refinement description / Category: pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: HCV NS3
B: HCV NS4a peptide
C: HCV NS3
D: HCV NS4a peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,0137
Polymers47,2554
Non-polymers7583
Water4,630257
1
A: HCV NS3
B: HCV NS4a peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,3204
Polymers23,6272
Non-polymers6932
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2680 Å2
ΔGint-51 kcal/mol
Surface area10090 Å2
MethodPISA
2
C: HCV NS3
D: HCV NS4a peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,6933
Polymers23,6272
Non-polymers651
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1690 Å2
ΔGint-47 kcal/mol
Surface area7780 Å2
MethodPISA
3


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6670 Å2
ΔGint-121 kcal/mol
Surface area15580 Å2
MethodPISA
Unit cell
Length a, b, c (Å)224.483, 224.483, 75.786
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number155
Space group name H-MH32
DetailsThe asymmetric unit contains a dimer of the NS3-NS4a complex. This is only the protease domain of NS3 and a peptide of NS4a. This dimeric structure is the result of the soultion structure of the domain. The full length NS3 contains a 3-domain helicase as well and would not have the same dimeric interfaces.

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Components

#1: Protein HCV NS3


Mass: 21233.225 Da / Num. of mol.: 2 / Fragment: Protease domain, UNP residues 1027-1207
Source method: isolated from a genetically manipulated source
Details: T7 epitope (MASMTGGQQMG) followed by HCV NS3 residues 1-181 the SGHHHHHH
Source: (gene. exp.) Hepatitis C virus subtype 1a / Strain: H77 Strain of genotype 1a / Gene: ns3 / Plasmid: pET-3a (Novagen) / Production host: Escherichia coli (E. coli) / Strain (production host): bl21(de3) / References: UniProt: Q9ELS8, UniProt: P26664*PLUS
#2: Protein/peptide HCV NS4a peptide


Mass: 2394.039 Da / Num. of mol.: 2 / Fragment: UNP residues 1678-1696 / Mutation: C22S / Source method: obtained synthetically
Details: Peptides were synthesized using Fmoc solid-phase chemistry on an ABI 431 synthesizer (Foster City, CA). Preloaded 2-chlorotrityl chloride resin or Wang resin was used for the solid phase ...Details: Peptides were synthesized using Fmoc solid-phase chemistry on an ABI 431 synthesizer (Foster City, CA). Preloaded 2-chlorotrityl chloride resin or Wang resin was used for the solid phase assembly of NS4A activator peptide. The sequence of the peptide was Lys-Lys-Gly-Ser-Val-Val-Ile-Val-Gly-Arg-Ile-Ile-Leu-Ser-Gly-Arg-Pro-Ala-Ile-Val-Pro-Lys-Lys-OH. Trifunctional residue sidechain protecting groups included tert-butyl for Ser, tert-butoxycarbonyl for Lys, and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl for Arg. Cleavage and sidechain deprotection was accomplished using 92.5% trifluoroacetic acid, with 2.5% each of water, ethanedithiol and triisopropylsilane for 2 hours. The peptide was purified by reversed phase HPLC. The peptide molecular weight was confirmed by electrospray ionization mass spectrometry.
References: UniProt: Q9ELS8, UniProt: P26664*PLUS
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-BE8 / [(1R)-2-cyclobutyl-1-({[(1R,2S,5S)-3-(N-{[(1S)-2,2-dimethyl-1-{[methyl(methylsulfonyl)amino]methyl}propyl]carbamoyl}-3-methyl-L-valyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-2-yl]carbonyl}amino)ethyl]boronic acid


Mass: 627.644 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C29H54BN5O7S
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 257 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.89 Å3/Da / Density % sol: 68.37 %
Crystal growMethod: vapor diffusion, hanging drop / pH: 5.6
Details: Crystallization was performed by the vapor diffusion method using hanging drops (4 L protein solution mixed with 4 L (0.75 - 1.00) M NaCl - 0.1 M MES - 0.1 M Na/K PO4, pH 5.6 - 6.2) ...Details: Crystallization was performed by the vapor diffusion method using hanging drops (4 L protein solution mixed with 4 L (0.75 - 1.00) M NaCl - 0.1 M MES - 0.1 M Na/K PO4, pH 5.6 - 6.2) suspended over 1 mL reservoir solutions of (1.25 - 1.50) M NaCl - 0.1 M MES - 0.1 M Na/K PO4 - 5 mM -mercaptoethanol, pH 5.6-6.2. The trays were set at 4oC for 5-7 days to control nucleation, followed by incubation for 3 weeks at 12oC to maximize crystal growth., VAPOR DIFFUSION, HANGING DROP, temperature 277, then 285K
Temp details: 277, then 285

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 17-ID / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Apr 7, 2004
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.15→50 Å / Num. all: 39911 / Num. obs: 28576 / % possible obs: 71.6 % / Observed criterion σ(I): 1 / Redundancy: 2.8 % / Rmerge(I) obs: 0.075 / Net I/σ(I): 13.5
Reflection shellResolution: 2.15→2.2 Å / Rmerge(I) obs: 0.288 / Mean I/σ(I) obs: 2 / Num. unique all: 2631 / % possible all: 20.6

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
X-PLORmodel building
X-PLOR98.1refinement
HKL-2000data reduction
SCALEPACKdata scaling
X-PLORphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: HCV NS3(1-181)S139A - NS4a pdb2o8m.ent

Resolution: 2.3→8 Å / Isotropic thermal model: anoistropic / Cross valid method: THROUGHOUT / σ(F): 1
RfactorNum. reflectionSelection details
Rfree0.256 2089 Random
Rwork0.18 --
all-25697 -
obs-20909 -
Refinement stepCycle: LAST / Resolution: 2.3→8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2727 0 45 257 3029
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.009
X-RAY DIFFRACTIONc_angle_deg2.019
X-RAY DIFFRACTIONc_improper_angle_d1.476
LS refinement shellResolution: 2.3→2.4 Å
RfactorNum. reflection% reflection
Rfree0.3443 103 -
Rwork0.2876 --
obs-972 40 %

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