Abstract:
:The 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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Pan Q,Shan Y,Yan Adoi
10.1021/bi2018688subject
Has Abstractpub_date
2012-06-26 00:00:00pages
5061-71issue
25eissn
0006-2960issn
1520-4995journal_volume
51pub_type
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