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- PDB-2xwy: Structure of MK-3281, a Potent Non-Nucleoside Finger-Loop Inhibit... -

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Basic information

Entry
Database: PDB / ID: 2xwy
TitleStructure of MK-3281, a Potent Non-Nucleoside Finger-Loop Inhibitor, in complex with the Hepatitis C Virus NS5B Polymerase
ComponentsRNA-DIRECTED RNA POLYMERASE
KeywordsTRANSFERASE / ALLOSTERIC INHIBITOR / NUCLEOTIDYLTRANSFERASE
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 / SH3 domain binding / nucleoside-triphosphate phosphatase ...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 / SH3 domain binding / 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 perinuclear region of cytoplasm / host cell endoplasmic reticulum membrane / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / RNA helicase / ribonucleoprotein complex / induction by virus of host autophagy / RNA-directed RNA polymerase / 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 / host cell plasma membrane / structural molecule activity / virion membrane / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane
Similarity search - Function
: / 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 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. / Reverse transcriptase/Diguanylate cyclase domain / DEAD box, Flavivirus / Flavivirus DEAD 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 / Helicase superfamily 1/2, ATP-binding domain / Reverse transcriptase/Diguanylate cyclase domain / Alpha-Beta Plaits / 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 / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-IB8 / : / Genome polyprotein
Similarity search - Component
Biological speciesHEPATITIS C VIRUS
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.53 Å
AuthorsDi Marco, S. / Baiocco, P.
CitationJournal: J.Med.Chem. / Year: 2011
Title: Discovery of (7R)-14-Cyclohexyl-7-{[2-(Dimethylamino)Ethyl] (Methyl)Amino}-7,8-Dihydro-6H-Indolo[1,2-E][1,5] Benzoxazocine -11-Carboxylic Acid (Mk-3281), a Potent and Orally Bioavailable ...Title: Discovery of (7R)-14-Cyclohexyl-7-{[2-(Dimethylamino)Ethyl] (Methyl)Amino}-7,8-Dihydro-6H-Indolo[1,2-E][1,5] Benzoxazocine -11-Carboxylic Acid (Mk-3281), a Potent and Orally Bioavailable Finger-Loop Inhibitor of the Hepatitis C Virus Ns5B Polymerase
Authors: Narjes, F. / Crescenzi, B. / Ferrara, M. / Habermann, J. / Colarusso, S. / Del Rosario Rico Ferreira, M. / Stansfield, I. / Mackay, A.C. / Conte, I. / Ercolani, C. / Zaramella, S. / Palumbi, ...Authors: Narjes, F. / Crescenzi, B. / Ferrara, M. / Habermann, J. / Colarusso, S. / Del Rosario Rico Ferreira, M. / Stansfield, I. / Mackay, A.C. / Conte, I. / Ercolani, C. / Zaramella, S. / Palumbi, M.C. / Meuleman, P. / Leroux-Roels, G. / Giuliano, C. / Fiore, F. / Di Marco, S. / Baiocco, P. / Koch, U. / Migliaccio, G. / Altamura, S. / Laufer, R. / De Francesco, R. / Rowley, M.
History
DepositionNov 6, 2010Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 22, 2010Provider: repository / Type: Initial release
Revision 1.1Oct 26, 2011Group: Database references / Version format compliance
Revision 1.2Apr 3, 2019Group: Data collection / Experimental preparation / Other
Category: exptl_crystal_grow / pdbx_database_proc / pdbx_database_status
Item: _exptl_crystal_grow.temp / _pdbx_database_status.recvd_author_approval
Revision 1.3Dec 20, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _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.pdbx_dist_value / _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_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_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-DIRECTED RNA POLYMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)60,3584
Polymers59,7731
Non-polymers5853
Water2,342130
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)66.983, 94.272, 95.145
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212

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Components

#1: Protein RNA-DIRECTED RNA POLYMERASE / NS5B / P68


Mass: 59772.711 Da / Num. of mol.: 1 / Fragment: NS5B, RESIDUES 2420-2955
Source method: isolated from a genetically manipulated source
Details: RESIDUES 2420-2955 OF HCV POLYPROTEIN / Source: (gene. exp.) HEPATITIS C VIRUS / Strain: ISOLATE BK / Variant: GENOTYPE 1B / Plasmid: PT7.7 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P26663, RNA-directed RNA polymerase
#2: Chemical ChemComp-IB8 / (7R)-14-cyclohexyl-7-{[2-(dimethylamino)ethyl](methyl)amino}-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic acid


Mass: 475.622 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C29H37N3O3
#3: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 130 / 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: 2.6 Å3/Da / Density % sol: 53 % / Description: NONE
Crystal growTemperature: 277 K / pH: 6
Details: SOAKING WITH COMPOUND MK3281 WAS PERFORMED AT 4 DEG C BY TRANSFERRING THE APO-NS5B CRYSTALS FOR 10 MIN IN A SOLUTION CONTAINING 100 MM 2-MORPHOLINOETHANESULFONIC ACID (MES), PH 6.0, 14% ...Details: SOAKING WITH COMPOUND MK3281 WAS PERFORMED AT 4 DEG C BY TRANSFERRING THE APO-NS5B CRYSTALS FOR 10 MIN IN A SOLUTION CONTAINING 100 MM 2-MORPHOLINOETHANESULFONIC ACID (MES), PH 6.0, 14% POLYETHYLENE GLYCOL (PEG) 8,000, 14% 2- PROPANOL, 2.5 MM TCEP, 10 MM MNCL2 AND 2.5 MM OF COMPOUND MK3281. THE CRYSTALS WERE THEN TRANSFERRED FOR 1 MIN TO A CRYO-PROTECTANT SOLUTION CONTAINING 100 MM MES PH 6.0, 14% POLYETHYLENE GLYCOL PEG 8,000, 14% 2-PROPANOL, 18% 2-METHYL-2,5 PENTANEDIOL (MPD), 2.5 MM TCEP, 5 MM MNCL2 AND 1.25 MM COMPOUND MK3281 AND THEN PLUNGED DIRECTLY INTO LIQUID NITROGEN.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID14-1 / Wavelength: 1.033
DetectorType: ADSC QUANTUM 210 / Detector: CCD
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.033 Å / Relative weight: 1
ReflectionResolution: 2.53→20 Å / Num. obs: 20692 / % possible obs: 99.7 % / Observed criterion σ(I): 1 / Redundancy: 3.9 % / Rmerge(I) obs: 0.09 / Net I/σ(I): 5.9
Reflection shellResolution: 2.53→2.67 Å / Redundancy: 3.8 % / Rmerge(I) obs: 0.53 / Mean I/σ(I) obs: 1.5 / % possible all: 99.7

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Processing

Software
NameVersionClassification
REFMAC5.5.0102refinement
MOSFLMdata reduction
SCALAdata scaling
AMoREphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1CSJ

1csj


Resolution: 2.53→95.35 Å / Cor.coef. Fo:Fc: 0.93 / Cor.coef. Fo:Fc free: 0.891 / SU B: 34.259 / SU ML: 0.35 / Cross valid method: THROUGHOUT / ESU R: 0.679 / ESU R Free: 0.366 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES WITH TLS ADDED. NO ELECTRON DENSITY WAS PRESENT FOR RESIDUES 15-35, 150- 152 AND 532-536 WHICH WERE THEREFORE EXCLUDED FROM THE ...Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES WITH TLS ADDED. NO ELECTRON DENSITY WAS PRESENT FOR RESIDUES 15-35, 150- 152 AND 532-536 WHICH WERE THEREFORE EXCLUDED FROM THE REFINEMENT. GLN 148, PRO 149 AND HIS 502 HAVE BEEN MODELED AS ALA DUE TO THE LACK OF ELECTRON DENSITY FOR SIDE CHAINS.
RfactorNum. reflection% reflectionSelection details
Rfree0.31016 1056 5.1 %RANDOM
Rwork0.23332 ---
obs0.23727 19605 99.63 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 54.851 Å2
Baniso -1Baniso -2Baniso -3
1-1.15 Å20 Å20 Å2
2---5.32 Å20 Å2
3---4.17 Å2
Refinement stepCycle: LAST / Resolution: 2.53→95.35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3911 0 37 130 4078
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.0224030
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.3491.9745472
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.7345504
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.96222.875160
X-RAY DIFFRACTIONr_dihedral_angle_3_deg18.53515675
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.261532
X-RAY DIFFRACTIONr_chiral_restr0.0850.2622
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.0213000
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.4681.52529
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it0.91624075
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it1.54331501
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it2.5044.51397
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.53→2.596 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.412 70 -
Rwork0.346 1384 -
obs--97.39 %
Refinement TLS params.Method: refined / Origin x: 24.5934 Å / Origin y: 27.8694 Å / Origin z: 26.8098 Å
111213212223313233
T0.2354 Å2-0.0467 Å2-0.0874 Å2-0.0728 Å2-0.0013 Å2--0.1385 Å2
L0.4446 °2-0.1955 °2-0.1715 °2-1.6144 °2-0.117 °2--0.6912 °2
S-0.0318 Å °0.1137 Å °-0.061 Å °-0.588 Å °0.0312 Å °0.2691 Å °0.0983 Å °-0.1139 Å °0.0006 Å °

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