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PDBsum entry 2o9a
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DNA binding protein
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PDB id
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2o9a
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References listed in PDB file
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Key reference
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Title
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Glyoxylate and pyruvate are antagonistic effectors of the escherichia coli iclr transcriptional regulator.
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Authors
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G.L.Lorca,
A.Ezersky,
V.V.Lunin,
J.R.Walker,
S.Altamentova,
E.Evdokimova,
M.Vedadi,
A.Bochkarev,
A.Savchenko.
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Ref.
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J Biol Chem, 2007,
282,
16476-16491.
[DOI no: ]
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PubMed id
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Abstract
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The Escherichia coli Isocitrate Lyase Regulator (IclR) regulates the expression
of the glyoxylate bypass operon (aceBAK). Founding member of a large family of
common fold transcriptional regulators - IclR comprises a DNA-binding domain
that interacts with the operator sequence and a C-terminal domain (C-IclR) that
binds a hitherto unknown small molecule. We screened a chemical library of over
150 metabolic scaffolds using a high-throughput protein stability assay to
identify molecules that bind IclR, and then tested the active compounds in in
vitro assays of operator binding. Glyoxylate and pyruvate, identified by this
method bound C-IclR domain with KDs of 0.9 +/- 0.2 mM and 156.2 +/- 7.9 mM, as
defined by isothermal titration calorimetry. Both compounds altered IclR
interactions with operator DNA in EMSA assays but showed an antagonistic effect.
Glyoxylate disrupted the formation of the IclR/operator complex in vitro by
favoring the inactive dimeric state of the protein while pyruvate increased the
binding of IclR to the aceBAK promoter by stabilizing the active tetrameric form
of the protein. Structures of the C-IclR domain alone and in complex with each
effector were determined at 2.3 A, confirming the binding of both molecules in
the effector recognition site previously characterized for the other
representative of the family, the E. coli AllR regulator. Site directed
mutagenesis demonstrated the importance of hydrophobic patch formed by Met146,
Leu154, Leu220, and Leu143 in interactions with effector molecules. In general,
our strategy of combining chemical screens with functional assays and structural
studies has uncovered two small molecules with antagonistic effects that
regulate the IclR-dependent transcription of the aceBAK operon.
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Figure 7.
FIGURE 7. Overall structure of C-IclR. A, schematic diagram
of a monomer of the IclR ligand binding domain in complex with
glyoxylate (PDB code 2O99). B, C-IclR interdomain interface
between two monomers in the (PDB code 2O99) structure. Loops are
colored green,  helices are red, and
 -strands are yellow.
Glyoxylate is represented as a stick figure in cyan. The N and C
termini are labeled.
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Figure 12.
FIGURE 12.
Functionalanalysisofkeyaminoacidsinvolvedininterdomaininteractionsandeffectorbinding.
The glyoxylate and pyruvate binding on mutant IclR proteins was
tested by EMSA. The name of the corresponding mutant protein and
its concentration used for binding is indicated above each
image. Glyoxylate and pyruvate were tested at 1 mM.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
16476-16491)
copyright 2007.
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