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Article: A region at the C-terminus of the Escherichia coli global transcription factor FNR negatively mediates its degradation by the ClpXP protease

TitleA region at the C-terminus of the Escherichia coli global transcription factor FNR negatively mediates its degradation by the ClpXP protease
Authors
Issue Date2012
PublisherAmerican Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistry
Citation
Biochemistry, 2012, v. 51 n. 25, p. 5061-5071 How to Cite?
AbstractThe anaerobic global regulator FNR from Escherichia coli is a [4Fe-4S] 2+ cluster-containing dimer that is inactivated by O 2 through disruption of the Fe-S cluster and conversion to the monomeric apoprotein. It was shown that apo-FNR is subject to ClpXP proteolysis, and two recognition sites, amino acids 5-11 and amino acids 249 and 250, are responsible for targeting FNR to the protease. However, how the exposure of these sites is mediated such that only apo-FNR is recognized by the ClpXP protease and is degraded in a regulated manner so that a sufficient and similar FNR level is maintained in both anaerobic and aerobic conditions is unknown. To investigate this, we performed three-alanine scanning on amino acids 2-19 and 236-250 that are in the proximity of the two ClpXP recognition sites, and their functions remain unknown. We found that three-alanine substitution of residues 239-241 (LAQ239-241A 3) and 242-244 (LAG242-244A 3) caused reduced FNR protein levels, transcription activities, and growth rates under anaerobic conditions. In vivo degradation assays demonstrated that these mutants were degraded significantly faster than the wild type (WT), and either deletion of clpXP or blocking the ClpXP recognition site of amino acids 249 and 250 stabilizes these proteins. Circular dichroism analysis revealed that introduction of LAQ239-241A 3 caused conformational changes with a significant loss of secondary structures in both WT and an O 2 stable FNR dimer, FNR D154A. We propose that the region of amino acids 239-244 plays a negative role in the proteolysis of FNR by promoting a structural fold that limits the exposure of the proximal ClpXP site to the protease. © 2012 American Chemical Society.
Persistent Identifierhttp://hdl.handle.net/10722/160581
ISSN
2015 Impact Factor: 2.876
2015 SCImago Journal Rankings: 1.769
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorPan, Qen_HK
dc.contributor.authorShan, Yen_HK
dc.contributor.authorYan, Aen_HK
dc.date.accessioned2012-08-16T06:14:46Z-
dc.date.available2012-08-16T06:14:46Z-
dc.date.issued2012en_HK
dc.identifier.citationBiochemistry, 2012, v. 51 n. 25, p. 5061-5071en_HK
dc.identifier.issn0006-2960en_HK
dc.identifier.urihttp://hdl.handle.net/10722/160581-
dc.description.abstractThe anaerobic global regulator FNR from Escherichia coli is a [4Fe-4S] 2+ cluster-containing dimer that is inactivated by O 2 through disruption of the Fe-S cluster and conversion to the monomeric apoprotein. It was shown that apo-FNR is subject to ClpXP proteolysis, and two recognition sites, amino acids 5-11 and amino acids 249 and 250, are responsible for targeting FNR to the protease. However, how the exposure of these sites is mediated such that only apo-FNR is recognized by the ClpXP protease and is degraded in a regulated manner so that a sufficient and similar FNR level is maintained in both anaerobic and aerobic conditions is unknown. To investigate this, we performed three-alanine scanning on amino acids 2-19 and 236-250 that are in the proximity of the two ClpXP recognition sites, and their functions remain unknown. We found that three-alanine substitution of residues 239-241 (LAQ239-241A 3) and 242-244 (LAG242-244A 3) caused reduced FNR protein levels, transcription activities, and growth rates under anaerobic conditions. In vivo degradation assays demonstrated that these mutants were degraded significantly faster than the wild type (WT), and either deletion of clpXP or blocking the ClpXP recognition site of amino acids 249 and 250 stabilizes these proteins. Circular dichroism analysis revealed that introduction of LAQ239-241A 3 caused conformational changes with a significant loss of secondary structures in both WT and an O 2 stable FNR dimer, FNR D154A. We propose that the region of amino acids 239-244 plays a negative role in the proteolysis of FNR by promoting a structural fold that limits the exposure of the proximal ClpXP site to the protease. © 2012 American Chemical Society.en_HK
dc.languageengen_US
dc.publisherAmerican Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistryen_HK
dc.relation.ispartofBiochemistryen_HK
dc.subject.meshConserved Sequenceen_HK
dc.subject.meshDown-Regulation - physiologyen_HK
dc.subject.meshEndopeptidase Clp - chemistry - physiologyen_HK
dc.subject.meshEscherichia coli Proteins - antagonists & inhibitors - chemistry - genetics - physiologyen_HK
dc.subject.meshIron-Sulfur Proteins - antagonists & inhibitors - chemistry - geneticsen_HK
dc.subject.meshPeptide Fragments - chemistry - physiologyen_HK
dc.subject.meshProteolysisen_HK
dc.subject.meshTranscription Factors - antagonists & inhibitors - chemistry - physiologyen_HK
dc.titleA region at the C-terminus of the Escherichia coli global transcription factor FNR negatively mediates its degradation by the ClpXP proteaseen_HK
dc.typeArticleen_HK
dc.identifier.emailYan, A: ayan8@hku.hken_HK
dc.identifier.authorityYan, A=rp00823en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1021/bi2018688en_HK
dc.identifier.pmid22656860-
dc.identifier.scopuseid_2-s2.0-84862846170en_HK
dc.identifier.hkuros203626en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-84862846170&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume51en_HK
dc.identifier.issue25en_HK
dc.identifier.spage5061en_HK
dc.identifier.epage5071en_HK
dc.identifier.eissn1520-4995-
dc.identifier.isiWOS:000305661800009-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridPan, Q=55262239500en_HK
dc.identifier.scopusauthoridShan, Y=55262302600en_HK
dc.identifier.scopusauthoridYan, A=8621667000en_HK

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