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[[Image: | ==Crystal structure of streptavidin mutant L124R (M1) complexed with biotinyl p-nitroanilide (BNI)== | ||
<StructureSection load='1rxh' size='340' side='right' caption='[[1rxh]], [[Resolution|resolution]] 2.90Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[1rxh]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/Streptomyces_avidinii Streptomyces avidinii]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1RXH OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1RXH FirstGlance]. <br> | |||
</td></tr><tr><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=BNI:5-(2-OXO-HEXAHYDRO-THIENO[3,4-D]IMIDAZOL-6-YL)-PENTANOIC+ACID+(4-NITRO-PHENYL)-AMIDE'>BNI</scene><br> | |||
<tr><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[1ij8|1ij8]], [[1i9h|1i9h]], [[1rxj|1rxj]], [[1rxk|1rxk]]</td></tr> | |||
<tr><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1rxh FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1rxh OCA], [http://www.rcsb.org/pdb/explore.do?structureId=1rxh RCSB], [http://www.ebi.ac.uk/pdbsum/1rxh PDBsum]</span></td></tr> | |||
<table> | |||
== Evolutionary Conservation == | |||
[[Image:Consurf_key_small.gif|200px|right]] | |||
Check<jmol> | |||
<jmolCheckbox> | |||
<scriptWhenChecked>select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/rx/1rxh_consurf.spt"</scriptWhenChecked> | |||
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked> | |||
<text>to colour the structure by Evolutionary Conservation</text> | |||
</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/chain_selection.php?pdb_ID=2ata ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Avidin enhances the hydrolysis of biotinyl p-nitrophenyl ester (BNP) under mild alkaline conditions, whereas streptavidin prevents hydrolysis of BNP up to pH 12. Recently, we imposed hydrolytic activity on streptavidin by rational mutagenesis, based on the molecular elements responsible for the hydrolysis by avidin. Three mutants were designed, whereby the desired features, the distinctive L124R point mutation (M1), the L3,4 loop replacement (M2), and the combined mutation (M3), were transferred from avidin to streptavidin. The crystal structures of the mutants, in complex with biotinyl p-nitroanilide (BNA), the stable amide analogue of BNP, were determined. The results demonstrate that the point mutation alone has little effect on hydrolysis, and BNA exhibits a conformation similar to that of streptavidin. Substitution of a lengthier L3,4 loop (from avidin to streptavidin), resulted in an open conformation, thus exposing the ligand to solvent. Moreover, the amide bond of BNA was flipped relative to that of the streptavidin and M1 complexes, thus deflecting the nitro group toward Lys-121. Consequently, the leaving group potential of the nitrophenyl group of BNP is increased, and M2 hydrolyzes BNP at pH values >8.5. To better emulate the hydrolytic potential of avidin, M3 was required. The combination of loop replacement and point mutation served to further increase the leaving group potential by interaction of the nitro group with Arg-124 and Lys-121. The information derived from this study may provide insight into the design of enzymes and transfer of desired properties among homologous proteins. | |||
Structural elements responsible for conversion of streptavidin to a pseudoenzyme.,Eisenberg-Domovich Y, Pazy Y, Nir O, Raboy B, Bayer EA, Wilchek M, Livnah O Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):5916-21. Epub 2004 Apr 12. PMID:15079055<ref>PMID:15079055</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
==See Also== | ==See Also== | ||
*[[Avidin|Avidin]] | *[[Avidin|Avidin]] | ||
== References == | |||
== | <references/> | ||
< | __TOC__ | ||
</StructureSection> | |||
[[Category: Streptomyces avidinii]] | [[Category: Streptomyces avidinii]] | ||
[[Category: Bayer, E A.]] | [[Category: Bayer, E A.]] | ||
Revision as of 23:29, 28 September 2014
Crystal structure of streptavidin mutant L124R (M1) complexed with biotinyl p-nitroanilide (BNI)Crystal structure of streptavidin mutant L124R (M1) complexed with biotinyl p-nitroanilide (BNI)
Structural highlights
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 PubMedAvidin enhances the hydrolysis of biotinyl p-nitrophenyl ester (BNP) under mild alkaline conditions, whereas streptavidin prevents hydrolysis of BNP up to pH 12. Recently, we imposed hydrolytic activity on streptavidin by rational mutagenesis, based on the molecular elements responsible for the hydrolysis by avidin. Three mutants were designed, whereby the desired features, the distinctive L124R point mutation (M1), the L3,4 loop replacement (M2), and the combined mutation (M3), were transferred from avidin to streptavidin. The crystal structures of the mutants, in complex with biotinyl p-nitroanilide (BNA), the stable amide analogue of BNP, were determined. The results demonstrate that the point mutation alone has little effect on hydrolysis, and BNA exhibits a conformation similar to that of streptavidin. Substitution of a lengthier L3,4 loop (from avidin to streptavidin), resulted in an open conformation, thus exposing the ligand to solvent. Moreover, the amide bond of BNA was flipped relative to that of the streptavidin and M1 complexes, thus deflecting the nitro group toward Lys-121. Consequently, the leaving group potential of the nitrophenyl group of BNP is increased, and M2 hydrolyzes BNP at pH values >8.5. To better emulate the hydrolytic potential of avidin, M3 was required. The combination of loop replacement and point mutation served to further increase the leaving group potential by interaction of the nitro group with Arg-124 and Lys-121. The information derived from this study may provide insight into the design of enzymes and transfer of desired properties among homologous proteins. Structural elements responsible for conversion of streptavidin to a pseudoenzyme.,Eisenberg-Domovich Y, Pazy Y, Nir O, Raboy B, Bayer EA, Wilchek M, Livnah O Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):5916-21. Epub 2004 Apr 12. PMID:15079055[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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