3fbd: Difference between revisions
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==Crystal structure of the nuclease domain of COLE7(D493Q mutant) in complex with an 18-BP duplex DNA== | ==Crystal structure of the nuclease domain of COLE7(D493Q mutant) in complex with an 18-BP duplex DNA== | ||
<StructureSection load='3fbd' size='340' side='right' caption='[[3fbd]], [[Resolution|resolution]] 2.90Å' scene=''> | <StructureSection load='3fbd' size='340' side='right'caption='[[3fbd]], [[Resolution|resolution]] 2.90Å' scene=''> | ||
== Structural highlights == | == Structural highlights == | ||
<table><tr><td colspan='2'>[[3fbd]] is a 6 chain structure with sequence from [ | <table><tr><td colspan='2'>[[3fbd]] is a 6 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3FBD OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3FBD FirstGlance]. <br> | ||
</td></tr><tr><td class="sblockLbl"><b>[[ | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.9Å</td></tr> | ||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=3fbd FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3fbd OCA], [https://pdbe.org/3fbd PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3fbd RCSB], [https://www.ebi.ac.uk/pdbsum/3fbd PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3fbd ProSAT]</span></td></tr> | |||
<tr><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[ | </table> | ||
<table> | == Function == | ||
[https://www.uniprot.org/uniprot/CEA7_ECOLX CEA7_ECOLX] This plasmid-coded bactericidal protein is an endonuclease active on both single- and double-stranded DNA but with undefined specificity. Colicins are polypeptide toxins produced by and active against E.coli and closely related bacteria. | |||
== Evolutionary Conservation == | == Evolutionary Conservation == | ||
[[Image:Consurf_key_small.gif|200px|right]] | [[Image:Consurf_key_small.gif|200px|right]] | ||
Check<jmol> | Check<jmol> | ||
<jmolCheckbox> | <jmolCheckbox> | ||
<scriptWhenChecked>select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/fb/3fbd_consurf.spt"</scriptWhenChecked> | <scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/fb/3fbd_consurf.spt"</scriptWhenChecked> | ||
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked> | <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked> | ||
<text>to colour the structure by Evolutionary Conservation</text> | <text>to colour the structure by Evolutionary Conservation</text> | ||
</jmolCheckbox> | </jmolCheckbox> | ||
</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/ | </jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=3fbd ConSurf]. | ||
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
It is of crucial importance to elucidate the underlying principles that govern the binding affinity and selectivity between proteins and DNA. Here we use the nuclease domain of Colicin E7 (nColE7) as a model system to generate redesigned nucleases with improved DNA-binding affinities. ColE7 is a bacterial toxin, bearing a nonspecific endonuclease domain with a preference for hydrolyzing DNA phosphodiester bonds at the 3'O-side after thymine and adenine; i.e., it prefers Thy and Ade at the -1 site. Using systematic computational screening, six nColE7 mutants were predicted to bind DNA with high affinity. Five of the redesigned single-point mutants were constructed and purified, and four mutants had a 3- to 5-fold higher DNA binding affinity than wild-type nColE7 as measured by fluorescence kinetic assays. Moreover, three of the designed mutants, D493N, D493Q, and D493R, digested DNA with an increased preference for guanine at +3 sites compared to the wild-type enzyme, as shown by DNA footprint assays. X-ray structure determination of the ColE7 mutant D493Q-DNA complex in conjunction with structural and free energy decomposition analyses provides a physical basis for the improved protein-DNA interactions: Replacing D493 at the protein-DNA interface with an amino acid residue that can maintain the native hydrogen bonds removes the unfavorable electrostatic repulsion between the negatively charged carboxylate and DNA phosphate groups. These results show that computational screening combined with biochemical, structural, and free energy analyses provide a useful means for generating redesigned nucleases with a higher DNA-binding affinity and altered sequence preferences in DNA cleavage. | |||
Redesign of high-affinity nonspecific nucleases with altered sequence preference.,Wang YT, Wright JD, Doudeva LG, Jhang HC, Lim C, Yuan HS J Am Chem Soc. 2009 Dec 2;131(47):17345-53. doi: 10.1021/ja907160r. PMID:19929021<ref>PMID:19929021</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 3fbd" style="background-color:#fffaf0;"></div> | |||
==See Also== | ==See Also== | ||
*[[Colicin|Colicin]] | *[[Colicin 3D structures|Colicin 3D structures]] | ||
== References == | |||
<references/> | |||
__TOC__ | __TOC__ | ||
</StructureSection> | </StructureSection> | ||
[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
[[Category: | [[Category: Large Structures]] | ||
[[Category: | [[Category: Doudeva LG]] | ||
[[Category: | [[Category: Wang YT]] | ||
[[Category: | [[Category: Yuan HS]] | ||
Latest revision as of 18:28, 1 November 2023
Crystal structure of the nuclease domain of COLE7(D493Q mutant) in complex with an 18-BP duplex DNACrystal structure of the nuclease domain of COLE7(D493Q mutant) in complex with an 18-BP duplex DNA
Structural highlights
FunctionCEA7_ECOLX This plasmid-coded bactericidal protein is an endonuclease active on both single- and double-stranded DNA but with undefined specificity. Colicins are polypeptide toxins produced by and active against E.coli and closely related bacteria. Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedIt is of crucial importance to elucidate the underlying principles that govern the binding affinity and selectivity between proteins and DNA. Here we use the nuclease domain of Colicin E7 (nColE7) as a model system to generate redesigned nucleases with improved DNA-binding affinities. ColE7 is a bacterial toxin, bearing a nonspecific endonuclease domain with a preference for hydrolyzing DNA phosphodiester bonds at the 3'O-side after thymine and adenine; i.e., it prefers Thy and Ade at the -1 site. Using systematic computational screening, six nColE7 mutants were predicted to bind DNA with high affinity. Five of the redesigned single-point mutants were constructed and purified, and four mutants had a 3- to 5-fold higher DNA binding affinity than wild-type nColE7 as measured by fluorescence kinetic assays. Moreover, three of the designed mutants, D493N, D493Q, and D493R, digested DNA with an increased preference for guanine at +3 sites compared to the wild-type enzyme, as shown by DNA footprint assays. X-ray structure determination of the ColE7 mutant D493Q-DNA complex in conjunction with structural and free energy decomposition analyses provides a physical basis for the improved protein-DNA interactions: Replacing D493 at the protein-DNA interface with an amino acid residue that can maintain the native hydrogen bonds removes the unfavorable electrostatic repulsion between the negatively charged carboxylate and DNA phosphate groups. These results show that computational screening combined with biochemical, structural, and free energy analyses provide a useful means for generating redesigned nucleases with a higher DNA-binding affinity and altered sequence preferences in DNA cleavage. Redesign of high-affinity nonspecific nucleases with altered sequence preference.,Wang YT, Wright JD, Doudeva LG, Jhang HC, Lim C, Yuan HS J Am Chem Soc. 2009 Dec 2;131(47):17345-53. doi: 10.1021/ja907160r. PMID:19929021[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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