Journal: 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
Deposition
Apr 4, 2014
Deposition site: PDBE / Processing site: PDBE
Revision 1.0
May 20, 2015
Provider: repository / Type: Initial release
Revision 1.1
Jul 15, 2015
Group: Database references / Other / Structure summary
SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "BA" IN EACH CHAIN ON SHEET RECORDS BELOW ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "BA" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 7-STRANDED BARREL THIS IS REPRESENTED BY A 8-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL.
Monochromator: SI(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
Wavelength: 0.91841 Å / Relative weight: 1
Reflection
Resolution: 2.53→80 Å / Num. obs: 18230 / % possible obs: 97.8 % / Observed criterion σ(I): -3 / Redundancy: 3.8 % / Biso Wilson estimate: 55.9 Å2 / Rmerge(I) obs: 0.08 / Net I/σ(I): 14.29
Reflection shell
Resolution: 2.53→2.68 Å / Redundancy: 3.3 % / Rmerge(I) obs: 0.66 / Mean I/σ(I) obs: 2.06 / % possible all: 93.6
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Processing
Software
Name
Version
Classification
REFMAC
5.8.0069
refinement
XDS
datareduction
Aimless
datascaling
PHASER
phasing
Refinement
Method to determine structure: MOLECULAR REPLACEMENT Starting model: DIMERIC PRV PROTEASE, YET TO BE DEPOSITED Resolution: 2.53→79.94 Å / Cor.coef. Fo:Fc: 0.949 / Cor.coef. Fo:Fc free: 0.912 / SU B: 21.997 / SU ML: 0.232 / Cross valid method: THROUGHOUT / ESU R: 0.423 / ESU R Free: 0.279 / Stereochemistry target values: MAXIMUM LIKELIHOOD Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. VALUES WITH TLS ADDED
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.25215
878
4.8 %
RANDOM
Rwork
0.19819
-
-
-
obs
0.20078
17352
97.75 %
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Solvent computation
Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.1 Å / Solvent model: MASK