2b0d: Difference between revisions

Revision as of 17:32, 21 February 2008

EcoRV Restriction Endonuclease/GAATTC/Ca2+

OverviewOverview

The crystal structure of EcoRV endonuclease bound to non-cognate DNA at 2.0 angstroms resolution shows that very small structural adaptations are sufficient to ensure the extreme sequence specificity characteristic of restriction enzymes. EcoRV bends its specific GATATC site sharply by 50 degrees into the major groove at the center TA step, generating unusual base-base interactions along each individual DNA strand. In the symmetric non-cognate complex bound to GAATTC, the center step bend is relaxed to avoid steric hindrance caused by the different placement of the exocyclic thymine methyl groups. The decreased base-pair unstacking in turn leads to small conformational rearrangements in the sugar-phosphate backbone, sufficient to destabilize binding of crucial divalent metal ions in the active site. A second crystal structure of EcoRV bound to the base-analog GAAUTC site shows that the 50 degrees center-step bend of the DNA is restored. However, while divalent metals bind at high occupancy in this structure, one metal ion shifts away from binding at the scissile DNA phosphate to a position near the 3'-adjacent phosphate group. This may explain why the 10(4)-fold attenuated cleavage efficiency toward GAATTC is reconstituted by less than tenfold toward GAAUTC. Examination of DNA binding and bending by equilibrium and stopped-flow florescence quenching and fluorescence resonance energy transfer (FRET) methods demonstrates that the capacity of EcoRV to bend the GAATTC non-cognate site is severely limited, but that full bending of GAAUTC is achieved at only a threefold reduced rate compared with the cognate complex. Together, the structural and biochemical data demonstrate the existence of distinct mechanisms for ensuring specificity at the bending and catalytic steps, respectively. The limited conformational rearrangements observed in the EcoRV non-cognate complex provide a sharp contrast to the extensive structural changes found in a non-cognate BamHI-DNA crystal structure, thus demonstrating a diversity of mechanisms by which restriction enzymes are able to achieve specificity.

About this StructureAbout this Structure

2B0D 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.

ReferenceReference

Non-cognate enzyme-DNA complex: structural and kinetic analysis of EcoRV endonuclease bound to the EcoRI recognition site GAATTC., Hiller DA, Rodriguez AM, Perona JJ, J Mol Biol. 2005 Nov 18;354(1):121-36. Epub 2005 Oct 3. PMID:16236314

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-[[Image:2b0d.gif|left|200px]]<br /><applet load="2b0d" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:2b0d.gif|left|200px]]<br /><applet load="2b0d" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="2b0d, resolution 2.0&Aring;" />
 '''EcoRV Restriction Endonuclease/GAATTC/Ca2+'''<br />
 ==Overview==
-The crystal structure of EcoRV endonuclease bound to non-cognate DNA at, 2.0 angstroms resolution shows that very small structural adaptations are, sufficient to ensure the extreme sequence specificity characteristic of, restriction enzymes. EcoRV bends its specific GATATC site sharply by 50, degrees into the major groove at the center TA step, generating unusual, base-base interactions along each individual DNA strand. In the symmetric, non-cognate complex bound to GAATTC, the center step bend is relaxed to, avoid steric hindrance caused by the different placement of the exocyclic, thymine methyl groups. The decreased base-pair unstacking in turn leads to, small conformational rearrangements in the sugar-phosphate backbone, sufficient to destabilize binding of crucial divalent metal ions in the, active site. A second crystal structure of EcoRV bound to the base-analog, GAAUTC site shows that the 50 degrees center-step bend of the DNA is, restored. However, while divalent metals bind at high occupancy in this, structure, one metal ion shifts away from binding at the scissile DNA, phosphate to a position near the 3'-adjacent phosphate group. This may, explain why the 10(4)-fold attenuated cleavage efficiency toward GAATTC is, reconstituted by less than tenfold toward GAAUTC. Examination of DNA, binding and bending by equilibrium and stopped-flow florescence quenching, and fluorescence resonance energy transfer (FRET) methods demonstrates, that the capacity of EcoRV to bend the GAATTC non-cognate site is severely, limited, but that full bending of GAAUTC is achieved at only a threefold, reduced rate compared with the cognate complex. Together, the structural, and biochemical data demonstrate the existence of distinct mechanisms for, ensuring specificity at the bending and catalytic steps, respectively. The, limited conformational rearrangements observed in the EcoRV non-cognate, complex provide a sharp contrast to the extensive structural changes found, in a non-cognate BamHI-DNA crystal structure, thus demonstrating a, diversity of mechanisms by which restriction enzymes are able to achieve, specificity.
+The crystal structure of EcoRV endonuclease bound to non-cognate DNA at 2.0 angstroms resolution shows that very small structural adaptations are sufficient to ensure the extreme sequence specificity characteristic of restriction enzymes. EcoRV bends its specific GATATC site sharply by 50 degrees into the major groove at the center TA step, generating unusual base-base interactions along each individual DNA strand. In the symmetric non-cognate complex bound to GAATTC, the center step bend is relaxed to avoid steric hindrance caused by the different placement of the exocyclic thymine methyl groups. The decreased base-pair unstacking in turn leads to small conformational rearrangements in the sugar-phosphate backbone, sufficient to destabilize binding of crucial divalent metal ions in the active site. A second crystal structure of EcoRV bound to the base-analog GAAUTC site shows that the 50 degrees center-step bend of the DNA is restored. However, while divalent metals bind at high occupancy in this structure, one metal ion shifts away from binding at the scissile DNA phosphate to a position near the 3'-adjacent phosphate group. This may explain why the 10(4)-fold attenuated cleavage efficiency toward GAATTC is reconstituted by less than tenfold toward GAAUTC. Examination of DNA binding and bending by equilibrium and stopped-flow florescence quenching and fluorescence resonance energy transfer (FRET) methods demonstrates that the capacity of EcoRV to bend the GAATTC non-cognate site is severely limited, but that full bending of GAAUTC is achieved at only a threefold reduced rate compared with the cognate complex. Together, the structural and biochemical data demonstrate the existence of distinct mechanisms for ensuring specificity at the bending and catalytic steps, respectively. The limited conformational rearrangements observed in the EcoRV non-cognate complex provide a sharp contrast to the extensive structural changes found in a non-cognate BamHI-DNA crystal structure, thus demonstrating a diversity of mechanisms by which restriction enzymes are able to achieve specificity.
 ==About this Structure==
-B0D 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://ispc.weizmann.ac.il/oca-bin/ocashort?id=2B0D OCA].
+B0D 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=2B0D OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Single protein]]
 [[Category: Type II site-specific deoxyribonuclease]]
-[[Category: Hiller, D.A.]]
+[[Category: Hiller, D A.]]
-[[Category: Perona, J.J.]]
+[[Category: Perona, J J.]]
-[[Category: Rodriguez, A.M.]]
+[[Category: Rodriguez, A M.]]
 [[Category: CA]]
 [[Category: indirect readout]]
@@ Line 24: / Line 24: @@
 [[Category: substrate specificity]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 08:29:49 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:32:52 2008''