6bcf: Difference between revisions
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==I-LtrI G183A bound to cognate substrate (pre-cleavage complex)== | |||
<StructureSection load='6bcf' size='340' side='right'caption='[[6bcf]], [[Resolution|resolution]] 2.92Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[6bcf]] is a 12 chain structure with sequence from [https://en.wikipedia.org/wiki/Leptographium_truncatum Leptographium truncatum]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6BCF OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=6BCF FirstGlance]. <br> | |||
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.92Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CA:CALCIUM+ION'>CA</scene></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=6bcf FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6bcf OCA], [https://pdbe.org/6bcf PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6bcf RCSB], [https://www.ebi.ac.uk/pdbsum/6bcf PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6bcf ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/C7SWF3_9PEZI C7SWF3_9PEZI] | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
LAGLIDADG homing endonucleases (meganucleases) are site-specific mobile endonucleases that can be adapted for genome-editing applications. However, one problem when reprogramming meganucleases on non-native substrates is indirect readout of DNA shape and flexibility at the central 4 bases where cleavage occurs. To understand how the meganuclease active site regulates DNA cleavage, we used functional selections and deep sequencing to profile the fitness landscape of 1600 I-LtrI and I-OnuI active site variants individually challenged with 67 substrates with central 4 base substitutions. The wild-type active site was not optimal for cleavage on many substrates, including the native I-LtrI and I-OnuI targets. Novel combinations of active site residues not observed in known meganucleases supported activity on substrates poorly cleaved by the wild-type enzymes. Strikingly, combinations of E or D substitutions in the two metal-binding residues greatly influenced cleavage activity, and E184D variants had a broadened cleavage profile. Analyses of I-LtrI E184D and the wild-type proteins co-crystallized with the non-cognate AACC central 4 sequence revealed structural differences that correlated with kinetic constants for cleavage of individual DNA strands. Optimizing meganuclease active sites to enhance cleavage of non-native central 4 target sites is a straightforward addition to engineering workflows that will expand genome-editing applications. | |||
Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases.,McMurrough TA, Brown CM, Zhang K, Hausner G, Junop MS, Gloor GB, Edgell DR Nucleic Acids Res. 2018 Oct 24. pii: 5144155. doi: 10.1093/nar/gky976. PMID:30357419<ref>PMID:30357419</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
[[Category: | <div class="pdbe-citations 6bcf" style="background-color:#fffaf0;"></div> | ||
[[Category: | == References == | ||
[[Category: | <references/> | ||
[[Category: | __TOC__ | ||
[[Category: | </StructureSection> | ||
[[Category: | [[Category: Large Structures]] | ||
[[Category: Leptographium truncatum]] | |||
[[Category: Brown C]] | |||
[[Category: Edgell DR]] | |||
[[Category: Gloor GB]] | |||
[[Category: Junop M]] | |||
[[Category: McMurrough TA]] | |||
[[Category: Zhang K]] | |||
Latest revision as of 17:38, 4 October 2023
I-LtrI G183A bound to cognate substrate (pre-cleavage complex)I-LtrI G183A bound to cognate substrate (pre-cleavage complex)
Structural highlights
FunctionPublication Abstract from PubMedLAGLIDADG homing endonucleases (meganucleases) are site-specific mobile endonucleases that can be adapted for genome-editing applications. However, one problem when reprogramming meganucleases on non-native substrates is indirect readout of DNA shape and flexibility at the central 4 bases where cleavage occurs. To understand how the meganuclease active site regulates DNA cleavage, we used functional selections and deep sequencing to profile the fitness landscape of 1600 I-LtrI and I-OnuI active site variants individually challenged with 67 substrates with central 4 base substitutions. The wild-type active site was not optimal for cleavage on many substrates, including the native I-LtrI and I-OnuI targets. Novel combinations of active site residues not observed in known meganucleases supported activity on substrates poorly cleaved by the wild-type enzymes. Strikingly, combinations of E or D substitutions in the two metal-binding residues greatly influenced cleavage activity, and E184D variants had a broadened cleavage profile. Analyses of I-LtrI E184D and the wild-type proteins co-crystallized with the non-cognate AACC central 4 sequence revealed structural differences that correlated with kinetic constants for cleavage of individual DNA strands. Optimizing meganuclease active sites to enhance cleavage of non-native central 4 target sites is a straightforward addition to engineering workflows that will expand genome-editing applications. Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases.,McMurrough TA, Brown CM, Zhang K, Hausner G, Junop MS, Gloor GB, Edgell DR Nucleic Acids Res. 2018 Oct 24. pii: 5144155. doi: 10.1093/nar/gky976. PMID:30357419[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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