2cxv: Difference between revisions
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[[Image:2cxv.gif|left|200px]]<br /><applet load="2cxv" size=" | [[Image:2cxv.gif|left|200px]]<br /><applet load="2cxv" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="2cxv, resolution 1.40Å" /> | caption="2cxv, resolution 1.40Å" /> | ||
'''Dual Modes of Modification of Hepatitis A Virus 3C Protease by a Serine-Derived betaLactone: Selective Crystallization and High-resolution Structure of the His-102 Adduct'''<br /> | '''Dual Modes of Modification of Hepatitis A Virus 3C Protease by a Serine-Derived betaLactone: Selective Crystallization and High-resolution Structure of the His-102 Adduct'''<br /> | ||
==Overview== | ==Overview== | ||
Hepatitis A virus (HAV) 3C proteinase is a member of the picornain | Hepatitis A virus (HAV) 3C proteinase is a member of the picornain cysteine proteases responsible for the processing of the viral polyprotein, a function essential for viral maturation and infectivity. This and its structural similarity to other 3C and 3C-like proteases make it an attractive target for the development of antiviral drugs. Previous solution NMR studies have shown that a Cys24Ser (C24S) variant of HAV 3C protein, which displays catalytic properties indistinguishable from the native enzyme, is irreversibly inactivated by N-benzyloxycarbonyl-l-serine-beta-lactone (1a) through alkylation of the sulfur atom at the active site Cys172. However, crystallization of an enzyme-inhibitor adduct from the reaction mixture followed by X-ray structural analysis shows only covalent modification of the epsilon2-nitrogen of the surface His102 by the beta-lactone with no reaction at Cys172. Re-examination of the heteronuclear multiple quantum coherence (HMQC) NMR spectra of the enzyme-inhibitor mixture indicates that dual modes of single covalent modification occur with a >/=3:1 ratio of S-alkylation of Cys172 to N-alkylation of His102. The latter product crystallizes readily, probably due to the interaction between the phenyl ring of the N-benzyloxycarbonyl (N-Cbz) moiety and a hydrophobic pocket of a neighboring protein molecule in the crystal. Furthermore, significant structural changes are observed in the active site of the 3C protease, which lead to the formation of a functional catalytic triad with Asp84 accepting one hydrogen bond from His44. Although the 3C protease modified at Cys172 is catalytically inactive, the singly modified His102 N(epsilon2)-alkylated protein displays a significant level of enzymatic activity, which can be further modified/inhibited by N-iodoacetyl-valine-phenylalanine-amide (IVF) (in solution and in crystal) or excessive amount of the same beta-lactone inhibitor (in solution). The success of soaking IVF into HAV 3C-1a crystals demonstrates the usefulness of this new crystal form in the study of enzyme-inhibitor interactions in the proteolytic active site. | ||
==About this Structure== | ==About this Structure== | ||
2CXV is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Hepatitis_a_virus Hepatitis a virus] with BBL as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Picornain_3C Picornain 3C], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.4.22.28 3.4.22.28] Full crystallographic information is available from [http:// | 2CXV is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Hepatitis_a_virus Hepatitis a virus] with <scene name='pdbligand=BBL:'>BBL</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Picornain_3C Picornain 3C], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.4.22.28 3.4.22.28] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2CXV OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Picornain 3C]] | [[Category: Picornain 3C]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Bergmann, E | [[Category: Bergmann, E M.]] | ||
[[Category: Cherney, M | [[Category: Cherney, M M.]] | ||
[[Category: Jain, R | [[Category: Jain, R P.]] | ||
[[Category: James, M | [[Category: James, M N.G.]] | ||
[[Category: Lall, M | [[Category: Lall, M S.]] | ||
[[Category: Vederas, J | [[Category: Vederas, J C.]] | ||
[[Category: Yin, J.]] | [[Category: Yin, J.]] | ||
[[Category: BBL]] | [[Category: BBL]] | ||
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[[Category: picornavirus]] | [[Category: picornavirus]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:53:30 2008'' | ||
Revision as of 17:53, 21 February 2008
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Dual Modes of Modification of Hepatitis A Virus 3C Protease by a Serine-Derived betaLactone: Selective Crystallization and High-resolution Structure of the His-102 Adduct
OverviewOverview
Hepatitis A virus (HAV) 3C proteinase is a member of the picornain cysteine proteases responsible for the processing of the viral polyprotein, a function essential for viral maturation and infectivity. This and its structural similarity to other 3C and 3C-like proteases make it an attractive target for the development of antiviral drugs. Previous solution NMR studies have shown that a Cys24Ser (C24S) variant of HAV 3C protein, which displays catalytic properties indistinguishable from the native enzyme, is irreversibly inactivated by N-benzyloxycarbonyl-l-serine-beta-lactone (1a) through alkylation of the sulfur atom at the active site Cys172. However, crystallization of an enzyme-inhibitor adduct from the reaction mixture followed by X-ray structural analysis shows only covalent modification of the epsilon2-nitrogen of the surface His102 by the beta-lactone with no reaction at Cys172. Re-examination of the heteronuclear multiple quantum coherence (HMQC) NMR spectra of the enzyme-inhibitor mixture indicates that dual modes of single covalent modification occur with a >/=3:1 ratio of S-alkylation of Cys172 to N-alkylation of His102. The latter product crystallizes readily, probably due to the interaction between the phenyl ring of the N-benzyloxycarbonyl (N-Cbz) moiety and a hydrophobic pocket of a neighboring protein molecule in the crystal. Furthermore, significant structural changes are observed in the active site of the 3C protease, which lead to the formation of a functional catalytic triad with Asp84 accepting one hydrogen bond from His44. Although the 3C protease modified at Cys172 is catalytically inactive, the singly modified His102 N(epsilon2)-alkylated protein displays a significant level of enzymatic activity, which can be further modified/inhibited by N-iodoacetyl-valine-phenylalanine-amide (IVF) (in solution and in crystal) or excessive amount of the same beta-lactone inhibitor (in solution). The success of soaking IVF into HAV 3C-1a crystals demonstrates the usefulness of this new crystal form in the study of enzyme-inhibitor interactions in the proteolytic active site.
About this StructureAbout this Structure
2CXV is a Single protein structure of sequence from Hepatitis a virus with as ligand. Active as Picornain 3C, with EC number 3.4.22.28 Full crystallographic information is available from OCA.
ReferenceReference
Dual modes of modification of hepatitis A virus 3C protease by a serine-derived beta-lactone: selective crystallization and formation of a functional catalytic triad in the active site., Yin J, Bergmann EM, Cherney MM, Lall MS, Jain RP, Vederas JC, James MN, J Mol Biol. 2005 Dec 9;354(4):854-71. Epub 2005 Oct 14. PMID:16288920
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