2pgb
From Proteopedia
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| - | + | {{STRUCTURE_2pgb| PDB=2pgb | SCENE= }} | |
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'''Inhibitor-free human thrombin mutant C191A-C220A''' | '''Inhibitor-free human thrombin mutant C191A-C220A''' | ||
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==Overview== | ==Overview== | ||
| - | + | Little is known on the role of disulfide bonds in the catalytic domain of serine proteases. The Cys-191-Cys-220 disulfide bond is located between the 190 strand leading to the oxyanion hole and the 220-loop that contributes to the architecture of the primary specificity pocket and the Na+ binding site in allosteric proteases. Removal of this bond in thrombin produces an approximately 100-fold loss of activity toward several chromogenic and natural substrates carrying Arg or Lys at P1. Na+ activation is compromised, and no fluorescence change can be detected in response to Na+ binding. A 1.54-A resolution structure of the C191A/C220A mutant in the free form reveals a conformation similar to the Na+-free slow form of wild type. The lack of disulfide bond exposes the side chain of Asp-189 to solvent, flips the backbone O atom of Gly-219, and generates disorder in portions of the 186 and 220 loops defining the Na+ site. This conformation, featuring perturbation of the Na+ site but with the active site accessible to substrate, offers a possible representation of the recently identified E* form of thrombin. Disorder in the 186 and 220 loops and the flip of Gly-219 are corrected by the active site inhibitor H-D-Phe-Pro-Arg-CH(2)Cl, as revealed by the 1.8-A resolution structure of the complex. We conclude that the Cys-191-Cys-220 disulfide bond confers stability to the primary specificity pocket by shielding Asp-189 from the solvent and orients the backbone O atom of Gly-219 for optimal substrate binding. In addition, the disulfide bond stabilizes the 186 and 220 loops that are critical for Na+ binding and activation. | |
==Disease== | ==Disease== | ||
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==Reference== | ==Reference== | ||
| - | + | Important role of the cys-191 cys-220 disulfide bond in thrombin function and allostery., Bush-Pelc LA, Marino F, Chen Z, Pineda AO, Mathews FS, Di Cera E, J Biol Chem. 2007 Sep 14;282(37):27165-70. Epub 2007 Jul 18. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/17636263 17636263] | |
[[Category: Homo sapiens]] | [[Category: Homo sapiens]] | ||
[[Category: Protein complex]] | [[Category: Protein complex]] | ||
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[[Category: Mathews, F S.]] | [[Category: Mathews, F S.]] | ||
[[Category: Pineda, A O.]] | [[Category: Pineda, A O.]] | ||
| - | [[Category: | + | [[Category: Hydrolase]] |
| - | + | [[Category: Serine protease]] | |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Wed Apr 30 13:30:20 2008'' |
Revision as of 10:30, 30 April 2008
Inhibitor-free human thrombin mutant C191A-C220A
Contents |
Overview
Little is known on the role of disulfide bonds in the catalytic domain of serine proteases. The Cys-191-Cys-220 disulfide bond is located between the 190 strand leading to the oxyanion hole and the 220-loop that contributes to the architecture of the primary specificity pocket and the Na+ binding site in allosteric proteases. Removal of this bond in thrombin produces an approximately 100-fold loss of activity toward several chromogenic and natural substrates carrying Arg or Lys at P1. Na+ activation is compromised, and no fluorescence change can be detected in response to Na+ binding. A 1.54-A resolution structure of the C191A/C220A mutant in the free form reveals a conformation similar to the Na+-free slow form of wild type. The lack of disulfide bond exposes the side chain of Asp-189 to solvent, flips the backbone O atom of Gly-219, and generates disorder in portions of the 186 and 220 loops defining the Na+ site. This conformation, featuring perturbation of the Na+ site but with the active site accessible to substrate, offers a possible representation of the recently identified E* form of thrombin. Disorder in the 186 and 220 loops and the flip of Gly-219 are corrected by the active site inhibitor H-D-Phe-Pro-Arg-CH(2)Cl, as revealed by the 1.8-A resolution structure of the complex. We conclude that the Cys-191-Cys-220 disulfide bond confers stability to the primary specificity pocket by shielding Asp-189 from the solvent and orients the backbone O atom of Gly-219 for optimal substrate binding. In addition, the disulfide bond stabilizes the 186 and 220 loops that are critical for Na+ binding and activation.
Disease
Known disease associated with this structure: Dysprothrombinemia OMIM:[176930], Hyperprothrombinemia OMIM:[176930], Hypoprothrombinemia OMIM:[176930]
About this Structure
2PGB is a Protein complex structure of sequences from Homo sapiens. Full crystallographic information is available from OCA.
Reference
Important role of the cys-191 cys-220 disulfide bond in thrombin function and allostery., Bush-Pelc LA, Marino F, Chen Z, Pineda AO, Mathews FS, Di Cera E, J Biol Chem. 2007 Sep 14;282(37):27165-70. Epub 2007 Jul 18. PMID:17636263 Page seeded by OCA on Wed Apr 30 13:30:20 2008
