2ge5
From Proteopedia
(New page: 200px<br /><applet load="2ge5" size="450" color="white" frame="true" align="right" spinBox="true" caption="2ge5, resolution 2.400Å" /> '''EcoRV Restriction E...) |
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| - | [[Image:2ge5.gif|left|200px]]<br /><applet load="2ge5" size=" | + | [[Image:2ge5.gif|left|200px]]<br /><applet load="2ge5" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="2ge5, resolution 2.400Å" /> | caption="2ge5, resolution 2.400Å" /> | ||
'''EcoRV Restriction Endonuclease C-terminal deletion mutant/GATATC/Ca2+'''<br /> | '''EcoRV Restriction Endonuclease C-terminal deletion mutant/GATATC/Ca2+'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The carboxy-terminal subdomains of the homodimeric EcoRV restriction | + | The carboxy-terminal subdomains of the homodimeric EcoRV restriction endonuclease each bear a net charge of +4 and are positioned on the inner concave surface of the 50 degree DNA bend that is induced by the enzyme. A complete kinetic and structural analysis of a truncated EcoRV mutant lacking these domains was performed to assess the importance of this diffuse charge in facilitating DNA binding, bending, and cleavage. At the level of formation of an enzyme-DNA complex, the association rate for the dimeric mutant enzyme was sharply decreased by 10(3)-fold, while the equilibrium dissociation constant was weakened by nearly 10(6)-fold compared with that of wild-type EcoRV. Thus, the C-terminal subdomains strongly stabilize the enzyme-DNA ground-state complex in which the DNA is known to be bent. Further, the extent of DNA bending as observed by fluorescence resonance energy transfer was also significantly decreased. The crystal structure of the truncated enzyme bound to DNA and calcium ions at 2.4 A resolution reveals that the global fold is preserved and suggests that a divalent metal ion crucial to catalysis is destabilized in the active site. This may explain the 100-fold decrease in the rate of metal-dependent phosphoryl transfer observed for the mutant. These results show that diffuse positive charge associated with the C-terminal subdomains of EcoRV plays a key role in DNA association, bending, and cleavage. |
==About this Structure== | ==About this Structure== | ||
| - | 2GE5 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with CA as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Type_II_site-specific_deoxyribonuclease Type II site-specific deoxyribonuclease], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.21.4 3.1.21.4] Full crystallographic information is available from [http:// | + | 2GE5 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=CA:'>CA</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Type_II_site-specific_deoxyribonuclease Type II site-specific deoxyribonuclease], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.21.4 3.1.21.4] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2GE5 OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Type II site-specific deoxyribonuclease]] | [[Category: Type II site-specific deoxyribonuclease]] | ||
| - | [[Category: Hiller, D | + | [[Category: Hiller, D A.]] |
| - | [[Category: Perona, J | + | [[Category: Perona, J J.]] |
[[Category: CA]] | [[Category: CA]] | ||
[[Category: protein-dna complex]] | [[Category: protein-dna complex]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:30:47 2008'' |
Revision as of 15:30, 21 February 2008
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EcoRV Restriction Endonuclease C-terminal deletion mutant/GATATC/Ca2+
Overview
The carboxy-terminal subdomains of the homodimeric EcoRV restriction endonuclease each bear a net charge of +4 and are positioned on the inner concave surface of the 50 degree DNA bend that is induced by the enzyme. A complete kinetic and structural analysis of a truncated EcoRV mutant lacking these domains was performed to assess the importance of this diffuse charge in facilitating DNA binding, bending, and cleavage. At the level of formation of an enzyme-DNA complex, the association rate for the dimeric mutant enzyme was sharply decreased by 10(3)-fold, while the equilibrium dissociation constant was weakened by nearly 10(6)-fold compared with that of wild-type EcoRV. Thus, the C-terminal subdomains strongly stabilize the enzyme-DNA ground-state complex in which the DNA is known to be bent. Further, the extent of DNA bending as observed by fluorescence resonance energy transfer was also significantly decreased. The crystal structure of the truncated enzyme bound to DNA and calcium ions at 2.4 A resolution reveals that the global fold is preserved and suggests that a divalent metal ion crucial to catalysis is destabilized in the active site. This may explain the 100-fold decrease in the rate of metal-dependent phosphoryl transfer observed for the mutant. These results show that diffuse positive charge associated with the C-terminal subdomains of EcoRV plays a key role in DNA association, bending, and cleavage.
About this Structure
2GE5 is a Single protein structure of sequence from Escherichia coli with as ligand. Active as Type II site-specific deoxyribonuclease, with EC number 3.1.21.4 Full crystallographic information is available from OCA.
Reference
Positively charged C-terminal subdomains of EcoRV endonuclease: contributions to DNA binding, bending, and cleavage., Hiller DA, Perona JJ, Biochemistry. 2006 Sep 26;45(38):11453-63. PMID:16981705
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