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- PDB-1dy9: Inhibition of the Hepatitis C Virus NS3/4A Protease. The Crystal ... -

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Basic information

Entry
Database: PDB / ID: 1dy9
TitleInhibition of the Hepatitis C Virus NS3/4A Protease. The Crystal Structures of Two Protease-Inhibitor Complexes (inhibitor I)
Components
  • NONSTRUCTURAL PROTEIN NS4A (P4)
  • PROTEASE/HELICASE NS3 (P70)
KeywordsHYDROLASE/HYDROLASE INHIBITOR / SERINE PROTEASE / NS3 / NS4A / HEPATITIS C VIRUS / PROTEASE INHIBITION / HYDROLASE-HYDROLASE INHIBITOR COMPLEX
Function / homology
Function and homology information


RNA stabilization / RNA folding chaperone / DNA/DNA annealing activity / RNA strand annealing 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 ...RNA stabilization / RNA folding chaperone / DNA/DNA annealing activity / RNA strand annealing 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 / protein-DNA complex / : / nucleoside-triphosphate phosphatase / protein complex oligomerization / monoatomic ion channel activity / viral nucleocapsid / clathrin-dependent endocytosis of virus by host cell / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / RNA helicase activity / host cell endoplasmic reticulum membrane / host cell perinuclear region of cytoplasm / RNA helicase / induction by virus of host autophagy / ribonucleoprotein complex / RNA-directed RNA polymerase / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / serine-type endopeptidase activity / fusion of virus membrane with host endosome membrane / viral envelope / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / host cell nucleus / virion attachment to host cell / host cell plasma membrane / virion membrane / structural molecule activity / ATP hydrolysis activity / proteolysis / DNA binding / RNA binding / zinc ion binding / ATP binding / membrane
Similarity search - Function
Thrombin, subunit H - #120 / Hepatitis C virus core protein, chain A superfamily / 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 ...Thrombin, subunit H - #120 / Hepatitis C virus core protein, chain A superfamily / 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 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 / 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 / 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 / Trypsin-like serine proteases / 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
BOC-GLU-LEU-FKI, PEPTIDE INHIBITOR / Chem-2ZF / Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesHEPATITIS C VIRUS
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsDi Marco, S. / Rizzi, M. / Volpari, C. / Walsh, M. / Narjes, F. / Colarusso, S. / De Francesco, R. / Matassa, V.G. / Sollazzo, M.
CitationJournal: J.Biol.Chem. / Year: 2000
Title: Inhibition of the Hepatitis C Virus Ns3/4A Protease the Crystal Structures of Two Protease-Inhibitor Complexes
Authors: Di Marco, S. / Rizzi, M. / Volpari, C. / Walsh, M. / Narjes, F. / Colarusso, S. / De Francesco, R. / Matassa, V.G. / Sollazzo, M.
History
DepositionJan 31, 2000Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 28, 2001Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Atomic model / Database references ...Atomic model / Database references / Derived calculations / Non-polymer description / Structure summary / Version format compliance
Revision 1.2Nov 30, 2012Group: Other
Revision 1.3May 8, 2019Group: Data collection / Derived calculations ...Data collection / Derived calculations / Experimental preparation / Other
Category: exptl_crystal_grow / pdbx_database_proc ...exptl_crystal_grow / pdbx_database_proc / pdbx_database_status / struct_biol / struct_conn
Item: _exptl_crystal_grow.method / _pdbx_database_status.recvd_author_approval / _struct_conn.pdbx_leaving_atom_flag
Revision 1.4Oct 9, 2019Group: Data collection / Other / Category: pdbx_database_status / reflns_shell
Item: _pdbx_database_status.status_code_sf / _reflns_shell.pdbx_Rsym_value
Revision 1.5Dec 6, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_conn_type / 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_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_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.ptnr2_label_seq_id / _struct_conn_type.id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Remark 700 SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS AC AND BC IN EACH CHAIN ON SHEET RECORDS ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS AC AND BC IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 6-STRANDED BARREL THIS IS REPRESENTED BY A 7-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: PROTEASE/HELICASE NS3 (P70)
B: PROTEASE/HELICASE NS3 (P70)
C: NONSTRUCTURAL PROTEIN NS4A (P4)
D: NONSTRUCTURAL PROTEIN NS4A (P4)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)43,9567
Polymers42,8714
Non-polymers1,0843
Water3,837213
1
A: PROTEASE/HELICASE NS3 (P70)
C: NONSTRUCTURAL PROTEIN NS4A (P4)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)21,9453
Polymers21,4362
Non-polymers5091
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4770 Å2
ΔGint-19.4 kcal/mol
Surface area10070 Å2
MethodPQS
2
B: PROTEASE/HELICASE NS3 (P70)
D: NONSTRUCTURAL PROTEIN NS4A (P4)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,0114
Polymers21,4362
Non-polymers5752
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4820 Å2
ΔGint-22.4 kcal/mol
Surface area10050 Å2
MethodPQS
Unit cell
Length a, b, c (Å)94.350, 94.350, 82.370
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number169
Space group name H-MP61
Noncrystallographic symmetry (NCS)NCS oper: (Code: given
Matrix: (0.3724, 0.92807, -0.0026), (0.92806, -0.37241, -0.00323), (-0.00396, -0.00121, -0.99999)
Vector: 12.21626, -5.75718, 14.19541)

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Components

#1: Protein PROTEASE/HELICASE NS3 (P70)


Mass: 19749.553 Da / Num. of mol.: 2 / Fragment: PROTEASE
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HEPATITIS C VIRUS (ISOLATE TAIWAN) / Strain: ISOLATE TAIWAN / Description: CDNA OF HEPATITIS C VIRUS / Gene: HCV / Plasmid: PT7-7 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: Q81755, UniProt: P26662*PLUS, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
#2: Protein/peptide NONSTRUCTURAL PROTEIN NS4A (P4)


Mass: 1686.097 Da / Num. of mol.: 2 / Fragment: RESIDUES 956-967 / Mutation: YES / Source method: obtained synthetically / Source: (synth.) HEPATITIS C VIRUS / References: UniProt: Q81755, UniProt: P26662*PLUS
#3: Chemical ChemComp-2ZF / N-(tert-butoxycarbonyl)-L-alpha-glutamyl-N-[(1R)-1-(carboxycarbonyl)-3,3-difluoropropyl]-L-leucinamide


Type: peptide-like, Peptide-like / Class: Inhibitor / Mass: 509.498 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H33F2N3O9 / References: BOC-GLU-LEU-FKI, PEPTIDE INHIBITOR
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 213 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsGENOME POLYPROTEIN, ID POLG_HCVJA_7, PROTEASE NS3 GENOME POLYPROTEIN, ID POLG_HCVJA_8, ...GENOME POLYPROTEIN, ID POLG_HCVJA_7, PROTEASE NS3 GENOME POLYPROTEIN, ID POLG_HCVJA_8, NONSTRUCTURAL PROTEIN THE SWISSPROT ENTRY POLG_HCVJA GIVES A FULL DESCRIPTION OF THE GENOME POLYPROTEIN FROM HEPATITIS C VIRUS (ISOLATE JAPANESE) THAT CONTAINS THE FOLLOWING MATURE PROTEINS PRODUCED BY POST-TRANSLATIONAL PROCESSING - CAPSID PROTEIN C (CORE PROTEIN) (P22); ENVELOPE GLYCOPROTEIN E1 (GP32) (GP35); ENVELOPE GLYCOPROTEIN E2 (GP68) (GP70) (NS1); PROTEIN P7; NONSTRUCTURAL PROTEIN NS2 (P21) (EC 3.4.22.*); PROTEASE/HELICASE NS3 (P70) (EC 3.4.21.*); NONSTRUCTURAL PROTEIN NS4A (P4); NONSTRUCTURAL PROTEIN NS4B (P27); NONSTRUCTURAL PROTEIN NS5A (P56); NONSTRUCTURAL PROTEIN NS5B (P66) (P70) (RNA-DIRECTED RNA POLYMERASE) (EC 2.7.7.48)]. HOWEVER, THE SWISSPROT ENTRY Q81755 (ISOLATE TAIWANESE) IS A 100% MATCH TO THE NS3 PROTEIN STUDIED HERE.

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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: 2.37 Å3/Da / Density % sol: 47.68 %
Crystal growMethod: vapor diffusion / pH: 5.1
Details: THE NS3 PROTEIN (1MG/ML) WAS INCUBATE AT 4C WITH THE NS4A COFACTOR PEPTIDE, CONTAINING A SOLUBIZING LYSINE TAG AT ITS N- AND C-TERMINI(KGSVVIVGRIILSGRK), AT A MOLAR RATIO OF 1:2 AND ...Details: THE NS3 PROTEIN (1MG/ML) WAS INCUBATE AT 4C WITH THE NS4A COFACTOR PEPTIDE, CONTAINING A SOLUBIZING LYSINE TAG AT ITS N- AND C-TERMINI(KGSVVIVGRIILSGRK), AT A MOLAR RATIO OF 1:2 AND CONCENTRATED TO 290 MICROMOLAR. NS3J/4A CRYSTALS, WITH A MAXIMUM SIZE OF 0.6 X 0.3 X 0.2 MM**3, WERE OBTAINED BY BOTH HANGING- AND SITTING-DROP VAPOUR DIFFUSION METHODS AFTER TWO WEEKS AT ROOM TEMPERATURE, WITH 3.4 M NACL, 10MM DTT, 0.1 M CITRATE BUFFER PH 5.1. THE TERNARY COMPLEX WITH INHIBITOR WAS PREPARED BY ADDING 5.0 MM OF INIBITOR TO CRYSTALS THAT WERE STABILISED IN 4.5 M NACL, 10 MM DTT, 0.1 M CITRATE BUFFER, PH 5.1.
Crystal grow
*PLUS
Method: vapor diffusion, hanging drop
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formula
13.4 M1reservoirNaCl
24.8 mMcyclohexyl-pentyl-beta-D-maltoside1reservoir
35 mMdithiothreitol1reservoir
40.02 %1reservoirNaN3
50.1 Mcitrate1reservoir
64.5 M1dropNaCl
710 mMdithiothreitol1drop
80.1 Mcitrate1drop

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID14-3 / Wavelength: 0.936
DetectorType: MARRESEARCH / Detector: CCD / Date: Feb 15, 1999 / Details: TOROIDAL MIRROR
RadiationMonochromator: SI(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.936 Å / Relative weight: 1
ReflectionResolution: 2.1→20 Å / Num. obs: 23960 / % possible obs: 98.2 % / Observed criterion σ(I): -3 / Redundancy: 2.8 % / Biso Wilson estimate: 37.4 Å2 / Rsym value: 0.035 / Net I/σ(I): 32
Reflection shellResolution: 2.1→2.14 Å / Redundancy: 2.1 % / Mean I/σ(I) obs: 3.2 / Rsym value: 0.15 / % possible all: 98.4
Reflection
*PLUS
Num. measured all: 66924 / Rmerge(I) obs: 0.035
Reflection shell
*PLUS
% possible obs: 98.4 % / Rmerge(I) obs: 0.15

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Processing

Software
NameClassification
REFMACrefinement
DENZOdata reduction
SCALEPACKdata scaling
AMoREphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1DY7

1dy7


Resolution: 2.1→20 Å / SU B: 3.62 / SU ML: 0.097 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.22 / ESU R Free: 0.21
RfactorNum. reflection% reflectionSelection details
Rfree0.275 1224 5 %RANDOM
Rwork0.213 ---
obs0.207 23960 98.2 %-
Displacement parametersBiso mean: 43.5 Å2
Refinement stepCycle: LAST / Resolution: 2.1→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2734 0 71 213 3018
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONp_bond_d0.010.02
X-RAY DIFFRACTIONp_angle_d0.030.04
X-RAY DIFFRACTIONp_angle_deg
X-RAY DIFFRACTIONp_planar_d0.030.05
X-RAY DIFFRACTIONp_hb_or_metal_coord
X-RAY DIFFRACTIONp_mcbond_it2.92
X-RAY DIFFRACTIONp_mcangle_it4.183
X-RAY DIFFRACTIONp_scbond_it3.772
X-RAY DIFFRACTIONp_scangle_it5.493
X-RAY DIFFRACTIONp_plane_restr0.0120.02
X-RAY DIFFRACTIONp_chiral_restr0.1250.15
X-RAY DIFFRACTIONp_singtor_nbd0.1930.3
X-RAY DIFFRACTIONp_multtor_nbd0.2780.3
X-RAY DIFFRACTIONp_xhyhbond_nbd
X-RAY DIFFRACTIONp_xyhbond_nbd0.2010.3
X-RAY DIFFRACTIONp_planar_tor6.77
X-RAY DIFFRACTIONp_staggered_tor20.515
X-RAY DIFFRACTIONp_orthonormal_tor
X-RAY DIFFRACTIONp_transverse_tor26.420
X-RAY DIFFRACTIONp_special_tor

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