Structural insights into the molecular mechanism of Escherichia coli SdiA, a quorum-sensing receptor

Abstract

Escherichia coli SdiA is a quorum-sensing (QS) receptor that responds to autoinducers produced by other bacterial species to control cell division and virulence. Crystal structures reveal that E. coli SdiA, which is composed of an N-terminal ligand-binding domain and a C-terminal DNA-binding domain (DBD), forms a symmetrical dimer. Although each domain shows structural similarity to other QS receptors, SdiA differs from them in the relative orientation of the two domains, suggesting that its ligand-binding and DNA-binding functions are independent. Consistently, in DNA gel-shift assays the binding affinity of SdiA for the ftsQP2 promoter appeared to be insensitive to the presence of autoinducers. These results suggest that autoinducers increase the functionality of SdiA by enhancing the protein stability rather than by directly affecting the DNA-binding affinity. Structural analyses of the ligand-binding pocket showed that SdiA cannot accommodate ligands with long acyl chains, which was corroborated by isothermal titration calorimetry and thermal stability analyses. The formation of an intersubunit disulfide bond that might be relevant to modulation of the DNA-binding activity was predicted from the proximal position of two Cys residues in the DBDs of dimeric SdiA. It was confirmed that the binding affinity of SdiA for the uvrY promoter was reduced under oxidizing conditions, which suggested the possibility of regulation of SdiA by multiple independent signals such as quorum-sensing inducers and the oxidation state of the cell.

Molecular Insights into the Impact of Oxidative Stress on the Quorum-Sensing Regulator Protein LasR

“…Staphylococcus aureus AgrA has been shown to form an intramolecular disulfide bond within its DBD when oxidized, leading to a reduction in DNA binding affinity (19). Additionally, Escherichia coli SdiA, a LuxR homolog like LasR, forms an intermolecular disulfide bond within its DBD, covalently linking two monomers and altering promoter binding affinity when oxidized with the addition of 2 mM H 2 O 2 in EMSAs (20). These studies provide additional evidence for a possible intersection between QS and oxidative sensing in bacterial regulator proteins.…”

A structural perspective on the mechanisms of quorum sensing activation in bacteria

“…Dimeric SdiA has two Cys residues in the DBD conferring inter-cysteine distance within the range of disulphide bond formation (5.7 Å), which suggests that an intersubunit disulphide bond may be relevant to modulation of DNAbinding activity (Kim et al 2014). Furthermore, the binding affinity of SdiA for the uvrY promoter and the transcriptional activity of SdiA may be reduced under oxidative conditions.…”

“…Nevertheless, several studies have reported that E. coli SdiA has some selectivity to AI, especially for ligands with short chain length. This may occur because the occluded residues Phe59 and Gln72 of the AI-binding cavity limit the chain length of the acyl moiety to eight carbon atoms (Kim et al 2014).…”

“…Currently, only TraR (Agrobacterium tumefaciens) (Vannini et al 2002), LasR (Pseudomonas aeruginosa) (Bottomley et al 2007), QscR (P. aeruginosa) (Lintz et al 2011), CviR (Chromobacteriumviolaceum) (Chen et al 2011) and SdiA (Escherichia coli) (Kim et al 2014) have had their three-dimensional structures determined (Table I). However, just TraR, QscR, CviR and SdiA have been crystallized as full-length proteins.…”

“…The DBD has four α-helices and is characterized by a typical HTH DNA-binding motif formed by helices α8 and α9 ( Fig. 1e) (Kim et al 2014). The overall folding of the SdiA LBD and DBD are well conserved when compared to other LuxR-type receptors, as opposed to the orientation of the two domains, which is significantly different.…”