PDBsum entry 2o9a

DNA binding protein

PDB id: 2o9a

Contents

Protein chains

182 a.a. *

Ligands

EDO ×4

PYR ×4

Waters ×835

* Residue conservation analysis

PDB id:

2o9a

Name:

DNA binding protein

Title:

The crystal structure of the e.Coli iclr c-terminal fragment in complex with pyruvate.

Structure:

Acetate operon repressor. Chain: a, b, c, d. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.80Å R-factor: 0.176 R-free: 0.232

Authors:

V.V.Lunin,A.Ezersky,E.Evdokimova,M.Kudritska,A.Savchenko

Key ref:

G.L.Lorca et al. (2007). Glyoxylate and pyruvate are antagonistic effectors of the Escherichia coli ICLR transcriptional regulator. J Biol Chem, 282, 16476-16491. PubMed id: 17426033 DOI: 10.1074/jbc.M610838200

Date:

13-Dec-06 Release date: 10-Apr-07

Protein chains

P16528  (ICLR_ECOLI) -  Transcriptional repressor IclR from Escherichia coli (strain K12)

Seq: 274 a.a.

Struc: 182 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

DOI no: 10.1074/jbc.M610838200

J Biol Chem 282:16476-16491 (2007)

PubMed id: 17426033

Glyoxylate and pyruvate are antagonistic effectors of the Escherichia coli ICLR transcriptional regulator.

G.L.Lorca, A.Ezersky, V.V.Lunin, J.R.Walker, S.Altamentova, E.Evdokimova, M.Vedadi, A.Bochkarev, A.Savchenko.

ABSTRACT

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.

Selected figure(s)

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.

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.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 16476-16491) copyright 2007.

Figures were selected by an automated process.