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2i9k

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[[Image:2i9k.gif|left|200px]]<br /><applet load="2i9k" size="350" color="white" frame="true" align="right" spinBox="true"
 
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caption="2i9k, resolution 2.65&Aring;" />
 
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'''Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI'''<br />
 
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==Overview==
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==Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI==
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Improved sequence specificity of the DNA cytosine methyltransferase HhaI was achieved by disrupting interactions at a hydrophobic interface between the active site of the enzyme and a highly conserved flexible loop. Transient fluorescence experiments show that mutations disrupting this interface destabilize the positioning of the extrahelical, "flipped" cytosine base within the active site. The ternary crystal structure of the F124A M.HhaI bound to cognate DNA and the cofactor analogue S-adenosyl-l-homocysteine shows an increase in cavity volume between the flexible loop and the core of the enzyme. This cavity disrupts the interface between the loop and the active site, thereby destabilizing the extrahelical target base. The favored partitioning of the base-flipped enzyme-DNA complex back to the base-stacked intermediate results in the mutant enzyme discriminating better than the wild-type enzyme against non-cognate sites. Building upon the concepts of kinetic proofreading and our understanding of M.HhaI, we describe how a 16-fold specificity enhancement achieved with a double mutation at the loop/active site interface is acquired through destabilization of intermediates prior to methyltransfer rather than disruption of direct interactions between the enzyme and the substrate for M.HhaI.
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<StructureSection load='2i9k' size='340' side='right'caption='[[2i9k]], [[Resolution|resolution]] 2.65&Aring;' scene=''>
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== Structural highlights ==
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<table><tr><td colspan='2'>[[2i9k]] is a 3 chain structure with sequence from [https://en.wikipedia.org/wiki/Haemophilus_haemolyticus Haemophilus haemolyticus]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2I9K OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=2I9K FirstGlance]. <br>
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</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.65&#8491;</td></tr>
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<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=SAH:S-ADENOSYL-L-HOMOCYSTEINE'>SAH</scene></td></tr>
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<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=2i9k FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2i9k OCA], [https://pdbe.org/2i9k PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=2i9k RCSB], [https://www.ebi.ac.uk/pdbsum/2i9k PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=2i9k ProSAT]</span></td></tr>
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</table>
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== Function ==
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[https://www.uniprot.org/uniprot/MTH1_HAEPH MTH1_HAEPH] This methylase recognizes the double-stranded sequence GCGC, causes specific methylation on C-2 on both strands, and protects the DNA from cleavage by the HhaI endonuclease.
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== Evolutionary Conservation ==
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[[Image:Consurf_key_small.gif|200px|right]]
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Check<jmol>
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<jmolCheckbox>
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<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/i9/2i9k_consurf.spt"</scriptWhenChecked>
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<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked>
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<text>to colour the structure by Evolutionary Conservation</text>
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</jmolCheckbox>
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</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=2i9k ConSurf].
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<div style="clear:both"></div>
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==About this Structure==
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==See Also==
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2I9K is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Haemophilus_haemolyticus Haemophilus haemolyticus] with <scene name='pdbligand=SAH:'>SAH</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2I9K OCA].
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*[[DNA methyltransferase 3D structures|DNA methyltransferase 3D structures]]
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__TOC__
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==Reference==
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</StructureSection>
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Engineered extrahelical base destabilization enhances sequence discrimination of DNA methyltransferase M.HhaI., Youngblood B, Shieh FK, De Los Rios S, Perona JJ, Reich NO, J Mol Biol. 2006 Sep 15;362(2):334-46. Epub 2006 Jul 21. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=16919299 16919299]
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[[Category: Haemophilus haemolyticus]]
[[Category: Haemophilus haemolyticus]]
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[[Category: Single protein]]
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[[Category: Large Structures]]
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[[Category: Perona, J J.]]
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[[Category: De Los Rios S]]
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[[Category: Reich, N O.]]
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[[Category: Perona JJ]]
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[[Category: Rios, S De Los.]]
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[[Category: Reich NO]]
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[[Category: Shieh, F K.]]
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[[Category: Shieh FK]]
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[[Category: Youngblood, B.]]
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[[Category: Youngblood B]]
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[[Category: SAH]]
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[[Category: phe124ala mutation in m hhai]]
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:50:23 2008''
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Current revision

Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI

PDB ID 2i9k

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