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Article: Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment

TitleFunctional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment
Authors
Issue Date2003
PublisherBioMed Central Ltd. The Journal's web site is located at http://www.biomedcentral.com/bmcstructbiol/
Citation
Bmc Structural Biology, 2003, v. 3, p. 1-20 How to Cite?
AbstractBackground: Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. Results: Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations. Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. Conclusions: The asymmetry uncovered here appears to be a common feature of oligomeric aminoacyl-tRNA synthetases, and may play an important functional role. We suggest a manner in which catalytic efficiency could be improved by LysU operating in an alternating sites mechanism.
Persistent Identifierhttp://hdl.handle.net/10722/48384
ISSN
2015 Impact Factor: 1.926
2015 SCImago Journal Rankings: 1.118
PubMed Central ID
References

 

DC FieldValueLanguage
dc.contributor.authorHughes, SJen_HK
dc.contributor.authorTanner, JAen_HK
dc.contributor.authorHindley, ADen_HK
dc.contributor.authorMiller, ADen_HK
dc.contributor.authorGould, IRen_HK
dc.date.accessioned2008-05-22T04:11:07Z-
dc.date.available2008-05-22T04:11:07Z-
dc.date.issued2003en_HK
dc.identifier.citationBmc Structural Biology, 2003, v. 3, p. 1-20en_HK
dc.identifier.issn1472-6807en_HK
dc.identifier.urihttp://hdl.handle.net/10722/48384-
dc.description.abstractBackground: Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. Results: Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations. Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. Conclusions: The asymmetry uncovered here appears to be a common feature of oligomeric aminoacyl-tRNA synthetases, and may play an important functional role. We suggest a manner in which catalytic efficiency could be improved by LysU operating in an alternating sites mechanism.en_HK
dc.format.extent1540844 bytes-
dc.format.extent2386 bytes-
dc.format.mimetypeapplication/pdf-
dc.format.mimetypetext/plain-
dc.languageengen_HK
dc.publisherBioMed Central Ltd. The Journal's web site is located at http://www.biomedcentral.com/bmcstructbiol/en_HK
dc.relation.ispartofBMC Structural Biologyen_HK
dc.rightsB M C Structural Biology. Copyright © BioMed Central Ltd.en_HK
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.subject.meshComputer Simulationen_HK
dc.subject.meshLysine-tRNA Ligase - chemistryen_HK
dc.subject.meshModels, Chemicalen_HK
dc.subject.meshThermodynamicsen_HK
dc.subject.meshAdenosine Triphosphate - chemistryen_HK
dc.titleFunctional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experimenten_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=1472-6807&volume=3&spage=5&epage=&date=2003&atitle=Functional+asymmetry+in+the+lysyl-tRNA+synthetase+explored+by+molecular+dynamics,+free+energy+calculations+and+experimenten_HK
dc.identifier.emailTanner, JA:jatanner@hku.hken_HK
dc.identifier.authorityTanner, JA=rp00495en_HK
dc.description.naturepublished_or_final_versionen_HK
dc.identifier.doi10.1186/1472-6807-3-5en_HK
dc.identifier.pmid12787471en_HK
dc.identifier.pmcidPMC165585-
dc.identifier.scopuseid_2-s2.0-3042572904en_HK
dc.identifier.hkuros84805-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-3042572904&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume3en_HK
dc.identifier.spage1en_HK
dc.identifier.epage20en_HK
dc.publisher.placeUnited Kingdomen_HK
dc.identifier.scopusauthoridHughes, SJ=36942170200en_HK
dc.identifier.scopusauthoridTanner, JA=35513993000en_HK
dc.identifier.scopusauthoridHindley, AD=36860704800en_HK
dc.identifier.scopusauthoridMiller, AD=7406230808en_HK
dc.identifier.scopusauthoridGould, IR=36707470300en_HK

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