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- PDB-3knx: HCV NS3 protease domain with P1-P3 macrocyclic ketoamide inhibitor -

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Basic information

Entry
Database: PDB / ID: 3knx
TitleHCV NS3 protease domain with P1-P3 macrocyclic ketoamide inhibitor
Components
  • HCV NS3 Protease
  • HCV NS4a peptide
KeywordsVIRAL PROTEIN / Hepatitis C Virus / NS3 Protease Domain / serine protease / macrocyclic ketoamide inhibitor / ATP-binding / Envelope protein / Helicase / Hydrolase / Membrane / Nucleotide-binding / RNA replication / Transmembrane
Function / homology
Function and homology information


host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated suppression of host TRAF-mediated signal transduction / symbiont-mediated transformation of host cell / 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 / channel activity ...host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated suppression of host TRAF-mediated signal transduction / symbiont-mediated transformation of host cell / 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 / channel activity / monoatomic ion transmembrane transport / viral nucleocapsid / clathrin-dependent endocytosis of virus by host cell / RNA helicase activity / host cell perinuclear region of cytoplasm / host cell endoplasmic reticulum membrane / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / symbiont-mediated activation of host autophagy / ribonucleoprotein complex / viral RNA genome replication / cysteine-type endopeptidase activity / serine-type endopeptidase activity / RNA-dependent RNA polymerase activity / : / 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 / proteolysis / RNA binding / zinc ion binding / ATP binding / 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, C-terminal domain / 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 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, C-terminal domain / 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 core protein, chain A superfamily / : / Hepatitis C virus non-structural protein NS4b / Hepatitis C virus capsid protein / Hepatitis C virus, Non-structural protein NS2 / 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 protein membrane anchor / Hepatitis C virus non-structural 5a zinc finger domain / Hepatitis C virus non-structural 5a domain 1b / NS3 RNA helicase, C-terminal helical domain / Hepacivirus nonstructural protein 2 (NS2) protease domain profile. / 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 / Hepatitis C virus NS3 protease / Hepacivirus/Pegivirus NS3 protease domain profile. / 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 / Thrombin, subunit H / Helicase superfamily 1/2, ATP-binding domain / 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
BETA-MERCAPTOETHANOL / Chem-JZT / Genome polyprotein
Similarity search - Component
Biological speciesHepatitis C virus subtype 1a
MethodX-RAY DIFFRACTION / FOURIER SYNTHESIS / Resolution: 2.65 Å
AuthorsVenkatraman, S. / Velazquez, F. / Wu, W. / Blackman, M. / Chen, K.X. / Bogen, S. / Nair, L. / Tong, X. / Chase, R. / Hart, A. ...Venkatraman, S. / Velazquez, F. / Wu, W. / Blackman, M. / Chen, K.X. / Bogen, S. / Nair, L. / Tong, X. / Chase, R. / Hart, A. / Agrawal, S. / Pichardo, J. / Prongay, A. / Cheng, K.-C. / Girijavallabhan, V. / Piwinski, J. / Shih, N.-Y. / Njoroge, F.G.
CitationJournal: J.Med.Chem. / Year: 2009
Title: Discovery and structure-activity relationship of P1-P3 ketoamide derived macrocyclic inhibitors of hepatitis C virus NS3 protease.
Authors: Venkatraman, S. / Velazquez, F. / Wu, W. / Blackman, M. / Chen, K.X. / Bogen, S. / Nair, L. / Tong, X. / Chase, R. / Hart, A. / Agrawal, S. / Pichardo, J. / Prongay, A. / Cheng, K.C. / ...Authors: Venkatraman, S. / Velazquez, F. / Wu, W. / Blackman, M. / Chen, K.X. / Bogen, S. / Nair, L. / Tong, X. / Chase, R. / Hart, A. / Agrawal, S. / Pichardo, J. / Prongay, A. / Cheng, K.C. / Girijavallabhan, V. / Piwinski, J. / Shih, N.Y. / Njoroge, F.G.
History
DepositionNov 12, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 27, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 13, 2021Group: Database references / Derived calculations
Category: database_2 / pdbx_struct_conn_angle ...database_2 / pdbx_struct_conn_angle / 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 / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _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 1.3Apr 3, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)48,1928
Polymers47,2554
Non-polymers9384
Water2,954164
1
A: HCV NS3 Protease
B: HCV NS4a peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,5005
Polymers23,6272
Non-polymers8733
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2910 Å2
ΔGint-55 kcal/mol
Surface area10000 Å2
MethodPISA
2
C: HCV NS3 Protease
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 area1710 Å2
ΔGint-47 kcal/mol
Surface area7670 Å2
MethodPISA
Unit cell
Length a, b, c (Å)223.587, 223.587, 75.282
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

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Protein / Protein/peptide , 2 types, 4 molecules ACBD

#1: Protein HCV NS3 Protease


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 and the His-tag (SGHHHHHH)
Source: (gene. exp.) Hepatitis C virus subtype 1a / Strain: H77 Strain of genotype 1a / Gene: NS3 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q9ELS8
#2: Protein/peptide HCV NS4a peptide


Mass: 2394.039 Da / Num. of mol.: 2 / Fragment: UNP residues 1678-1696 / Mutation: C28S / 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

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Non-polymers , 4 types, 168 molecules

#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-JZT / (2R)-2-{(3S,13S,16aS,17aR,17bS)-13-[({(1S)-1-[(4,4-dimethyl-2,6-dioxopiperidin-1-yl)methyl]-2,2-dimethylpropyl}carbamoyl)amino]-17,17-dimethyl-1,14-dioxooctadecahydro-2H-cyclopropa[3,4]pyrrolo[1,2-a][1,4]diazacyclohexadecin-3-yl}-2-hydroxy-N-prop-2-en-1-ylethanamide


Mass: 728.961 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C39H64N6O7
#5: Chemical ChemComp-BME / BETA-MERCAPTOETHANOL


Mass: 78.133 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6OS
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 164 / 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: 3.83 Å3/Da / Density % sol: 67.9 %
Crystal growTemperature: 285 K / Method: 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

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU200 / Wavelength: 1.54 Å
DetectorType: RIGAKU RAXIS IV / Detector: IMAGE PLATE / Date: Apr 1, 2004
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 2.6→50 Å / Num. all: 22223 / Num. obs: 18845 / % possible obs: 84.8 % / Observed criterion σ(I): 3 / Redundancy: 4.1 % / Rmerge(I) obs: 0.089 / Net I/σ(I): 12.8
Reflection shellResolution: 2.6→2.66 Å / Rmerge(I) obs: 0.395 / Mean I/σ(I) obs: 1.5 / Num. unique all: 1074 / % possible all: 13.5

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Processing

Software
NameVersionClassification
CrystalCleardata collection
X-PLORmodel building
X-PLOR98.1refinement
HKL-2000data reduction
SCALEPACKdata scaling
X-PLORphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: HCV NS3(Ser139A)-NS4a peptide

Resolution: 2.65→8 Å / Isotropic thermal model: Isotropic / Cross valid method: THROUGHOUT / σ(I): 3 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.263 1305 -random
Rwork0.169 ---
obs-13172 9.9 %-
Refinement stepCycle: LAST / Resolution: 2.65→8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2724 0 58 164 2946
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONx_bond_d0.008
X-RAY DIFFRACTIONx_angle_d1.948
X-RAY DIFFRACTIONx_improper_angle_d1.543
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkRefine-IDNum. reflection obs% reflection obs (%)
2.65-2.770.3033280.2994X-RAY DIFFRACTION27110.8
2.77-2.910.3631660.2721X-RAY DIFFRACTION79831.8
2.91-3.080.34441240.2271X-RAY DIFFRACTION143957.8
3.08-3.30.28621730.2081X-RAY DIFFRACTION179070.9
3.3-3.60.25472090.1765X-RAY DIFFRACTION197578.7
3.6-4.060.23692310.155X-RAY DIFFRACTION216085.8

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