The X-ray structure of the Southampton norovirus 3C protease linked to an active site-directed inhibitor at 1.7°A resolution
The X-ray structure of the Southampton norovirus 3C protease linked to an active site-directed inhibitor at 1.7°A resolution
The gene encoding the 3C-protease from Southampton Norovirus (SV) was cloned and expressed using a recombinant strain of Escherichia coli. The resulting recombinant protease self-excised from the primary translation product and was purified to homogeneity in milligramme quantities. Substrate specificity of the protease was probed using a colourimetric assay in the form of a series of chromogenic peptides consisting of a yellow chromophore, p-nitroaniline, linked to the C-terminus of a substrate derived peptide chain of between 3 and 6 residues in length mimicking residues of the natural ORF-1 polyprotein substrate P1 to P6 residues.
Crystals of the SV 3C-protease (SV3CP) were obtained and an X-ray structural solution was sought, initially using molecular replacement and subsequently, by incorporating selenomethionine in place of the five methionine residues as a phase reference. A structural solution was finally achieved by MAD after the selenomethionine enzyme had been modified using a synthetic peptide linked to a Michael acceptor inhibitor, Ac-Glu-Phe-Gin-Leu-Gin-propenyl-ethyl-ester. This inhibitor specifically and irreversibly modified the active site cysteine 139 with the amino acids Glu, Phe, Leu and Gin occupying the S5, S4, S3, S2 and S1 binding sites, respectively. Two other active site amino acid residues, histidine 30 and glutamate 54 are positioned near cysteine 139 indicating the presence of a triad of catalytic residues. The X-ray structure at 1.75Å resolution allows the interactions of the inhibitor’s peptide portion to be defined at the SV3CP active site. Such information may be useful in further development of the Michael acceptor inhibitor or in the design of future prophylactic therapeutically viable agents to tackle Noroviral infection.
University of Southampton
Hussey, Robert John
c4b0cdab-0b32-4eb2-9c00-a07422b12edf
2007
Hussey, Robert John
c4b0cdab-0b32-4eb2-9c00-a07422b12edf
Hussey, Robert John
(2007)
The X-ray structure of the Southampton norovirus 3C protease linked to an active site-directed inhibitor at 1.7°A resolution.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The gene encoding the 3C-protease from Southampton Norovirus (SV) was cloned and expressed using a recombinant strain of Escherichia coli. The resulting recombinant protease self-excised from the primary translation product and was purified to homogeneity in milligramme quantities. Substrate specificity of the protease was probed using a colourimetric assay in the form of a series of chromogenic peptides consisting of a yellow chromophore, p-nitroaniline, linked to the C-terminus of a substrate derived peptide chain of between 3 and 6 residues in length mimicking residues of the natural ORF-1 polyprotein substrate P1 to P6 residues.
Crystals of the SV 3C-protease (SV3CP) were obtained and an X-ray structural solution was sought, initially using molecular replacement and subsequently, by incorporating selenomethionine in place of the five methionine residues as a phase reference. A structural solution was finally achieved by MAD after the selenomethionine enzyme had been modified using a synthetic peptide linked to a Michael acceptor inhibitor, Ac-Glu-Phe-Gin-Leu-Gin-propenyl-ethyl-ester. This inhibitor specifically and irreversibly modified the active site cysteine 139 with the amino acids Glu, Phe, Leu and Gin occupying the S5, S4, S3, S2 and S1 binding sites, respectively. Two other active site amino acid residues, histidine 30 and glutamate 54 are positioned near cysteine 139 indicating the presence of a triad of catalytic residues. The X-ray structure at 1.75Å resolution allows the interactions of the inhibitor’s peptide portion to be defined at the SV3CP active site. Such information may be useful in further development of the Michael acceptor inhibitor or in the design of future prophylactic therapeutically viable agents to tackle Noroviral infection.
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Published date: 2007
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Local EPrints ID: 466499
URI: http://eprints.soton.ac.uk/id/eprint/466499
PURE UUID: 4ee6c349-bff5-414b-89d8-2e180e9305df
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Date deposited: 05 Jul 2022 05:25
Last modified: 16 Mar 2024 20:44
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Author:
Robert John Hussey
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