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Title: Structure of the SARS coronavirus main proteinase as an active C{sub 2} crystallographic dimer

Abstract

An orthorhombic crystal form of the SARS CoV main proteinase diffracting to a resolution of 1.9 Å is reported. The conformation of residues in the catalytic site indicates an active enzyme. The 34 kDa main proteinase (M{sup pro}) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV M{sup pro} is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pH 6.5 in the orthorhombic space group P2{sub 1}2{sub 1}2 that diffract to a resolution of 1.9 Å. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies.

Authors:
; ; ;  [1];  [2];  [1]
  1. School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore)
  2. Institute of Molecular and Cell Biology (Singapore)
Publication Date:
OSTI Identifier:
22356178
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 61; Journal Issue: Pt 11; Other Information: PMCID: PMC1978130; PMID: 16511208; PUBLISHER-ID: sw5004; OAI: oai:pubmedcentral.nih.gov:1978130; Copyright (c) International Union of Crystallography 2005; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALS; DIMERS; MONOMERS; PROCESSING; RESOLUTION; SPACE GROUPS

Citation Formats

Xu, Ting, Ooi, Amy, Lee, Hooi Chen, Wilmouth, Rupert, Liu, Ding Xiang, and Lescar, Julien, E-mail: julien@ntu.edu.sg. Structure of the SARS coronavirus main proteinase as an active C{sub 2} crystallographic dimer. United Kingdom: N. p., 2005. Web. doi:10.1107/S1744309105033257.
Xu, Ting, Ooi, Amy, Lee, Hooi Chen, Wilmouth, Rupert, Liu, Ding Xiang, & Lescar, Julien, E-mail: julien@ntu.edu.sg. Structure of the SARS coronavirus main proteinase as an active C{sub 2} crystallographic dimer. United Kingdom. doi:10.1107/S1744309105033257.
Xu, Ting, Ooi, Amy, Lee, Hooi Chen, Wilmouth, Rupert, Liu, Ding Xiang, and Lescar, Julien, E-mail: julien@ntu.edu.sg. Tue . "Structure of the SARS coronavirus main proteinase as an active C{sub 2} crystallographic dimer". United Kingdom. doi:10.1107/S1744309105033257.
@article{osti_22356178,
title = {Structure of the SARS coronavirus main proteinase as an active C{sub 2} crystallographic dimer},
author = {Xu, Ting and Ooi, Amy and Lee, Hooi Chen and Wilmouth, Rupert and Liu, Ding Xiang and Lescar, Julien, E-mail: julien@ntu.edu.sg},
abstractNote = {An orthorhombic crystal form of the SARS CoV main proteinase diffracting to a resolution of 1.9 Å is reported. The conformation of residues in the catalytic site indicates an active enzyme. The 34 kDa main proteinase (M{sup pro}) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV M{sup pro} is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pH 6.5 in the orthorhombic space group P2{sub 1}2{sub 1}2 that diffract to a resolution of 1.9 Å. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies.},
doi = {10.1107/S1744309105033257},
journal = {Acta Crystallographica. Section F},
number = Pt 11,
volume = 61,
place = {United Kingdom},
year = {Tue Nov 01 00:00:00 EST 2005},
month = {Tue Nov 01 00:00:00 EST 2005}
}
  • The SARS coronavirus main proteinase (SARS CoV main proteinase) is required for the replication of the severe acute respiratory syndrome coronavirus (SARS CoV), the virus that causes SARS. One function of the enzyme is to process viral polyproteins. The active form of the SARS CoV main proteinase is a homodimer. In the literature, estimates of the monomer-dimer equilibrium dissociation constant, K{sub D}, have varied more than 650000-fold, from <1 nM to more than 200 {mu}M. Because of these discrepancies and because compounds that interfere with activation of the enzyme by dimerization may be potential antiviral agents, we investigated the monomer-dimermore » equilibrium by three different techniques: small-angle X-ray scattering, chemical cross-linking, and enzyme kinetics. Analysis of small-angle X-ray scattering data from a series of measurements at different SARS CoV main proteinase concentrations yielded K{sub D} values of 5.8 {+-} 0.8 {mu}M (obtained from the entire scattering curve), 6.5 {+-} 2.2 {mu}M (obtained from the radii of gyration), and 6.8 {+-} 1.5 {mu}M (obtained from the forward scattering). The K{sub D} from chemical cross-linking was 12.7 {+-} 1.1 {mu}M, and from enzyme kinetics, it was 5.2 {+-} 0.4 {mu}M. While each of these three techniques can present different, potential limitations, they all yielded similar K{sub D} values.« less
  • A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (16-(R), (R)-N-(4-(tert-butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Pubchem CID: 46897844). But, unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, 16-(R) is a noncovalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multicomponent Ugi reaction was utilized to rapidly explore structure–activity relationships within S1', S1, and S2enzyme binding pockets. Moreover, the X-ray structure of SARS-CoV 3CLpromore » bound with 16-(R) was instrumental in guiding subsequent rounds of chemistry optimization. 16-(R) provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a noncovalent mechanism of action.« less
  • This communication reports the synthesis and crystal structure of the first actinide phosphoylide complex, which possesses an unusual coordination number and exhibits a unique mode of ylide bonding. Synthesis was accomplished by the addition of Cp/sub 3/UCl to Li(CH/sub 2/)/sub 2/P(C/sub 6/H/sub 5/)/sub 2/ in a 1 : 2 molar ratio at -50/sup 0/C in diethyl ether followed by slow recrystallization of the product over a period of several days at room temperature. Intensity data using molybdenum-potassium alpha radiation were collected using an autodiffractometer indicating that the compound belongs in the space group P2/sub 1//c with a = 12.676(8)A, bmore » = 14.462(8)A, c = 25.837(25)A, ..beta.. = 124.43(5)A and z = 4. Structure was solved by heavy-atom techniques and refined by full-matrix least square methods, which employed anisotropic thermal parameters for the uranium and phosphorus atoms and isotropic thermal parameters for the carbon atoms. The molecule is a biscyclopentadienyluranium dimer bridged by phosphoylide ligands. The resulting ..mu..-carbon bridge is unique in actinide chemistry. Average bond lengths, bond angles, and a perspective view of the compound are presented.« less
  • The chemistry of (..mu..-H)/sub 2/Rh/sub 2/(P(0-i-C/sub 3/H/sub 7/)/sub 3/)/sub 4/ with carbon monoxide is described. One equivalent of carbon monoxide irreversibly converted the dimer to (..mu..-H)/sub 2/Rh/sub 2/(..mu..-CO)(P(0-i-C/sub 3/H/sub 7/)/sub 3/)/sub 4/. The latter transformed slowly in solution to generate Rh/sub 2/(..mu..-CO)/sub 2/(P(0-i-C/sub 3/H/sub 7/)/sub 3/)/sub 4/ and a second species presumed to be a hydride. The 30-electron Rh/sub 2/(..mu..-CO)/sub 2/(P(0-i-C/sub 3/H/sub 7/)/sub 3/)/sub 4/ dimer reacted reversibly with carbon monoxide to give Rh/sub 2/(..mu..-CO)/sub 2/(CO)/sub 2/(P(0-i-C/sub 3/H/sub 7/)/sub 3/)/sub 4/; no fragmentation to mononuclear metal complexes in this reaction system was observed up to 70/sup 0/C. Excess carbon monoxidemore » did however elicit a fragmentation of the 30-electron complex, (..mu..-H)/sub 2/Rh/sub 2/(..mu..-CO)(P(0-i-C/sub 3/H/sub 7/)/sub 3/)/sub 4/. Reaction was rapid to give HRh(CO)(P(0-i-C/sub 3/H/sub 7/)/sub 3/)/sub 3/ and HRh(CO)/sub 2/(P(0-i-C/sub 3/H/sub 7/)/sub 3/)/sub 2/. The chemical and dynamic solution properties of these two mononuclear species is described; the properties of HRh(CO)(P(0-i-C/sub 3/H/sub 7/)/sub 3/)/sub 3/ diverge sharply for uranium and 16 other elements in sediments, and for uranium and 9 other elements in ground water. Mass spectrometry results are given for helium in ground water. Field measurements and observations are reported for each site. Analytical data and field measurements are presented in tables and maps. Uranium concentrations in the sediments which were above detection limits ranged from 0.10 t 51.2 ppM. The mean of the logarithms of the uranium concentrations was 0.53. A group of high uranium concentrations occurs near the junctions of quadrangles AB, AC, BB, a 200 mK. In case 2), x-ray studies of isotopic phase separation in /sup 3/He--/sup 4/He bcc solids were carried out by B. A. Fraass.« less
  • Treatment of a methanol solution of Rh/sub 2/(CO)/sub 4/(..mu..-Cl)/sub 2/ with bis((dimethylphosphino)methyl)methylphosphine, dmmm, in dichloromethane followed by precipitation with sodium tetraphenylborate yields yellow (Rh/sub 2/(..mu..dmmm)/sub 2/(CO/sub 2/)(BPh/sub 4/)/sub 2/. The structure of this salt has been determined by X-ray crystallography. It crystallizes from acetone/ethylether in the orthorhombic space group Pn2/sup 1/m (bca of No. 31) with 2 molecules/unit cell of dimensions a = 11.590 (2), b = 12.516 (2), and c = 22.329 (4) A at 140 K. Least-squares refinement of 213 parameters using 2845 reflections yielded R = 0.057. the structure of the cation consists of a four-coordinate rhodiummore » (terminal CO, two CH/sub 3/P(CH/sub 2/)/sub 2/, and the other Rh as ligands) connected to a six-coordinate rhodium (terminal CO, four CH/sub 2/P(CH/sub 3/)/sub 2/, and the other Rh as ligands (through a dative Rh-Rh bond, which is 2.777 (1) A long. The coordination about the six-coordinate rhodium is distorted so that one trans P-Rh-P units nearly linear (173.9 (2)/sup 0/) and the other is decidedly bent (146.1 (2)/sup 0/). This distortion is caused by the packing of the methyl and methylene carbons around the OC-Rh-Rh-CO unit. The /sup 31/P NMR spectrum is temperature-dependent. In acetone solution at -75 /sup 0/C the three phosphorus environments are clearly resolved while warming causes broadening and approach to coalescence of the two terminal phosphorus resonances. 28 references, 6 figures, 3 tables.« less