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- PDB-4v08: Inhibited dimeric pseudorabies virus protease pUL26N at 2 A resolution -

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Basic information

Entry
Database: PDB / ID: 4v08
TitleInhibited dimeric pseudorabies virus protease pUL26N at 2 A resolution
ComponentsUL26
KeywordsVIRAL PROTEIN / ASSEMBLIN / UL26 / UL26P / SERINE PROTEASE / PROTEASE / SUID / PRV / HERPES / HERPES VIRUS
Function / homology
Function and homology information


assemblin / nuclear capsid assembly / viral release from host cell / host cell cytoplasm / serine-type endopeptidase activity / host cell nucleus / proteolysis / identical protein binding
Similarity search - Function
Serine Protease, Human Cytomegalovirus Protease; Chain A / Herpesvirus/Caudovirus protease domain / Peptidase S21 / Herpesvirus protease superfamily / Assemblin (Peptidase family S21) / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
DIISOPROPYL PHOSPHONATE / Capsid scaffolding protein
Similarity search - Component
Biological speciesSUID HERPESVIRUS 1
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.03 Å
AuthorsZuehlsdorf, M. / Werten, S. / Palm, G.J. / Hinrichs, W.
CitationJournal: PLoS Pathog / Year: 2015
Title: Dimerization-Induced Allosteric Changes of the Oxyanion-Hole Loop Activate the Pseudorabies Virus Assemblin pUL26N, a Herpesvirus Serine Protease.
Authors: Martin Zühlsdorf / Sebastiaan Werten / Barbara G Klupp / Gottfried J Palm / Thomas C Mettenleiter / Winfried Hinrichs /
Abstract: Herpesviruses encode a characteristic serine protease with a unique fold and an active site that comprises the unusual triad Ser-His-His. The protease is essential for viral replication and as such ...Herpesviruses encode a characteristic serine protease with a unique fold and an active site that comprises the unusual triad Ser-His-His. The protease is essential for viral replication and as such constitutes a promising drug target. In solution, a dynamic equilibrium exists between an inactive monomeric and an active dimeric form of the enzyme, which is believed to play a key regulatory role in the orchestration of proteolysis and capsid assembly. Currently available crystal structures of herpesvirus proteases correspond either to the dimeric state or to complexes with peptide mimetics that alter the dimerization interface. In contrast, the structure of the native monomeric state has remained elusive. Here, we present the three-dimensional structures of native monomeric, active dimeric, and diisopropyl fluorophosphate-inhibited dimeric protease derived from pseudorabies virus, an alphaherpesvirus of swine. These structures, solved by X-ray crystallography to respective resolutions of 2.05, 2.10 and 2.03 Å, allow a direct comparison of the main conformational states of the protease. In the dimeric form, a functional oxyanion hole is formed by a loop of 10 amino-acid residues encompassing two consecutive arginine residues (Arg136 and Arg137); both are strictly conserved throughout the herpesviruses. In the monomeric form, the top of the loop is shifted by approximately 11 Å, resulting in a complete disruption of the oxyanion hole and loss of activity. The dimerization-induced allosteric changes described here form the physical basis for the concentration-dependent activation of the protease, which is essential for proper virus replication. Small-angle X-ray scattering experiments confirmed a concentration-dependent equilibrium of monomeric and dimeric protease in solution.
History
DepositionSep 11, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 15, 2015Provider: repository / Type: Initial release
Revision 1.1Jul 22, 2015Group: Database references
Revision 2.0Apr 11, 2018Group: Atomic model / Data collection / Derived calculations
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / pdbx_nonpoly_scheme / pdbx_struct_conn_angle / pdbx_struct_sheet_hbond / struct_conf / struct_conn / struct_sheet / struct_sheet_order / struct_sheet_range / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site_anisotrop.pdbx_auth_seq_id / _pdbx_nonpoly_scheme.asym_id / _pdbx_nonpoly_scheme.auth_seq_num / _pdbx_nonpoly_scheme.ndb_seq_num / _pdbx_nonpoly_scheme.pdb_seq_num / _pdbx_nonpoly_scheme.pdb_strand_id / _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_symmetry / _pdbx_struct_conn_angle.ptnr2_auth_seq_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_symmetry / _pdbx_struct_conn_angle.value / _pdbx_struct_sheet_hbond.range_1_auth_atom_id / _pdbx_struct_sheet_hbond.range_1_auth_comp_id / _pdbx_struct_sheet_hbond.range_1_auth_seq_id / _pdbx_struct_sheet_hbond.range_1_label_atom_id / _pdbx_struct_sheet_hbond.range_1_label_comp_id / _pdbx_struct_sheet_hbond.range_1_label_seq_id / _pdbx_struct_sheet_hbond.range_2_auth_atom_id / _pdbx_struct_sheet_hbond.range_2_auth_comp_id / _pdbx_struct_sheet_hbond.range_2_auth_seq_id / _pdbx_struct_sheet_hbond.range_2_label_atom_id / _pdbx_struct_sheet_hbond.range_2_label_comp_id / _pdbx_struct_sheet_hbond.range_2_label_seq_id / _pdbx_struct_sheet_hbond.sheet_id / _struct_conf.pdbx_PDB_helix_id / _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_symmetry / _struct_sheet.id / _struct_sheet_order.sheet_id / _struct_sheet_range.sheet_id / _struct_site.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id / _struct_site.pdbx_num_residues
Revision 2.1Jan 10, 2024Group: Data collection / Database references ...Data collection / Database references / 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
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf
Revision 2.2Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: UL26
B: UL26
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,7548
Polymers53,2912
Non-polymers4636
Water4,738263
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3860 Å2
ΔGint-43.8 kcal/mol
Surface area19490 Å2
MethodPISA
Unit cell
Length a, b, c (Å)52.062, 76.226, 111.055
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS oper: (Code: given
Matrix: (0.09145, 0.02409, 0.9955), (0.1703, -0.9854, 0.008192), (0.9811, 0.1688, -0.09421)
Vector: -27.38, -61.5, 36.52)

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Components

#1: Protein UL26 / PSEUDORABIES VIRUS PROTEASE


Mass: 26645.357 Da / Num. of mol.: 2 / Fragment: RESIDUES 1-224
Source method: isolated from a genetically manipulated source
Details: N-TERMINAL (HIS)6-TAG WITH THROMBIN-LINKER / Source: (gene. exp.) SUID HERPESVIRUS 1 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q83417, assemblin
#2: Chemical ChemComp-DFP / DIISOPROPYL PHOSPHONATE


Mass: 166.155 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H15O3P
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 263 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY
Sequence detailsASSEMBLIN-PART CRYSTALLIZED (RES. 1-224)

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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: 2.07 Å3/Da / Density % sol: 40.63 %
Description: NO PHASING WAS NECESSARY SINCE THE CRYSTAL OF THE MODEL AND THE ONE USED FOR THIS DATASET WERE ISOMORPHOUS
Crystal growpH: 8
Details: PROTEIN SOLUTION WAS INCUBATED WITH 5 MM DFP FOR 1 HOUR, THEN CRYSTALLIZED FROM 0.1 M TRIS/HCL PH 8, 0.2 M MGCL2, 20% PEG 8000

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.91841
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Apr 30, 2014 / Details: MIRRORS
RadiationMonochromator: SI(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.91841 Å / Relative weight: 1
ReflectionResolution: 2.03→62.8 Å / Num. obs: 29319 / % possible obs: 99.9 % / Observed criterion σ(I): -3 / Redundancy: 6.5 % / Biso Wilson estimate: 35 Å2 / Rmerge(I) obs: 0.11 / Net I/σ(I): 13.08
Reflection shellResolution: 2.03→2.14 Å / Redundancy: 6.3 % / Rmerge(I) obs: 0.78 / Mean I/σ(I) obs: 2.2 / % possible all: 99.4

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Processing

Software
NameVersionClassification
REFMAC5.8.0073refinement
XDSdata reduction
Aimlessdata scaling
REFMACphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 4V07

4v07


Resolution: 2.03→62.85 Å / Cor.coef. Fo:Fc: 0.965 / Cor.coef. Fo:Fc free: 0.935 / SU B: 9.813 / SU ML: 0.137 / Cross valid method: THROUGHOUT / ESU R: 0.181 / ESU R Free: 0.174 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES WITH TLS ADDED, RESIDUES 1,46,218 OF CHAIN A AND RESIDUES 19,84,114,169,218 OF ...Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES WITH TLS ADDED, RESIDUES 1,46,218 OF CHAIN A AND RESIDUES 19,84,114,169,218 OF CHAIN B MODELED AS ALA AND RESIDUES 156 AND 169 FROM CHAIN A SHORTENED DUE TO INSUFFICIENT ELECTRON DENSITY, TAG AND THROMBIN-LINKER OF BOTH CHAINS AND RESIDUES 115-119 AND 219-224 OF CHAIN A ARE DISORDERED
RfactorNum. reflection% reflectionSelection details
Rfree0.23432 1433 4.9 %RANDOM
Rwork0.17028 ---
obs0.17346 27886 99.87 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 35.46 Å2
Baniso -1Baniso -2Baniso -3
1-1.72 Å20 Å20 Å2
2---0.71 Å20 Å2
3----1.01 Å2
Refinement stepCycle: LAST / Resolution: 2.03→62.85 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3306 0 24 263 3593
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0160.0193396
X-RAY DIFFRACTIONr_bond_other_d0.0010.023289
X-RAY DIFFRACTIONr_angle_refined_deg1.7631.9884624
X-RAY DIFFRACTIONr_angle_other_deg0.87937503
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.9035436
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.65821.781146
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.86115512
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.0341542
X-RAY DIFFRACTIONr_chiral_restr0.1020.2533
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.0213852
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02768
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_it1.8472.1761747
X-RAY DIFFRACTIONr_mcbond_other1.8462.1751746
X-RAY DIFFRACTIONr_mcangle_it2.8093.2482179
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it2.5822.5111649
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
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: 2.029→2.082 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.284 82 -
Rwork0.26 2047 -
obs--98.66 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.47741.0489-1.37871.172-1.27713.35490.13120.02310.24940.16020.06030.2719-0.1408-0.1119-0.19160.03980.00860.03810.01120.01770.0822-14.072-35.294839.0007
21.99010.7593-0.0561.4004-0.13751.2783-0.08220.0561-0.1302-0.22440.0984-0.11070.03390.0435-0.01620.1241-0.02490.01310.01780.00320.03129.5084-28.875713.4219
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 300
2X-RAY DIFFRACTION2B1 - 300

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