Structural basis for promoter DNA recognition by the response regulator OmpR

Highlights

• Crystal structures of E. coli OmpR DNA-binding domain were determined in apo and DNA-bound states.

• OmpR DNA-binding domain forms a unique domain-swapped dimer.

• OmpR DNA-binding domain shows intermolecular interactions in solution.

• Phosphorylation may only enhance but not be required for OmpR–DNA binding.

Abstract

OmpR, a response regulator of the EnvZ/OmpR two-component system (TCS), controls the reciprocal regulation of two porin proteins, OmpF and OmpC, in bacteria. During signal transduction, OmpR (OmpR-FL) undergoes phosphorylation at its conserved Asp residue in the N-terminal receiver domain (OmpRn) and recognizes the promoter DNA from its C-terminal DNA-binding domain (OmpRc) to elicit an adaptive response. Apart from that, OmpR regulates many genes in Escherichia coli and is important for virulence in several pathogens. However, the molecular mechanism of the regulation and the structural basis of OmpR–DNA binding is still not fully clear. In this study, we presented the crystal structure of OmpRc in complex with the F1 region of the ompF promoter DNA from E. coli. Our structural analysis suggested that OmpRc binds to its cognate DNA as a homodimer, only in a head-to-tail orientation. Also, the OmpRc apo-form showed a unique domain-swapped crystal structure under different crystallization conditions. Biophysical experimental data, such as NMR, fluorescent polarization and thermal stability, showed that inactive OmpR-FL (unphosphorylated) could bind to promoter DNA with a weaker binding affinity as compared with active OmpR-FL (phosphorylated) or OmpRc, and also confirmed that phosphorylation may only enhance DNA binding. Furthermore, the dimerization interfaces in the OmpRc–DNA complex structure identified in this study provide an opportunity to understand the regulatory role of OmpR and explore the potential for this “druggable” target.

Introduction

Survival of all organisms in nature depends on their ability to sense and respond to their surrounding environment. The two-component system (TCS) is one of the predominant signalling mechanisms found in various prokaryotes that responds to environmental stresses for transmitting signals inside cells (Chang and Stewart, 1998, Groisman, 2001, Parkinson and Kofoid, 1992). In Escherichia coli, the EnvZ/OmpR TCS is a well-studied signalling system responsible for the reciprocal regulation of two genes, ompF and ompC, in response to the osmolarity conditions (Russo and Silhavy, 1991, Vanalphen and Lugtenberg, 1977, Yoshida et al., 2006). Both porin proteins transcribed from these two genes predominate in the outer membrane at different osmolarity: OmpF at low osmolarity and OmpC at high osmolarity.

In the EnvZ/OmpR TCS, EnvZ acts as a transmembrane histidine kinase (HK) that becomes autophosphorylated at its conserved histidine residue upon sensing the signal (Roberts et al., 1994), then transfers the phosphoryl group to OmpR. In a typical TCS mechanism, the HK protein detects environmental changes through its sensory domain and becomes auto-phosphorylated at a conserved His residue at its cytoplasmic domain. However, the cytoplasmic domain of EnvZ can sense the intracellular signal and become activated (Foo et al., 2015, Wang et al., 2012). OmpR is the response regulator (RR) of this system that undergoes phosphorylation at its conserved Asp residue in the N-terminal receiver domain (Delgado et al., 1993) and binds to DNA from its C-terminal DNA-binding domain, thereby resulting in signal transduction. At low osmolarity, phosphorylated OmpR activates the transcription of OmpF protein; whereas, at high osmolarity, it activates the transcription of OmpC protein and represses that of OmpF (Forst and Inouye, 1988, Kenney et al., 1995, Mizuno and Mizushima, 1990). The cellular level of phosphorylated OmpR is controlled by the kinase and phosphatase activities of EnvZ, thereby regulating the transcriptional activity of OmpR.

In addition to involvement in osmotic stress, the EnvZ/OmpR TCS has a regulatory role in controlling the transcription of numerous genes in E. coli (Oshima et al., 2002). Recent studies showed the involvement of OmpR in regulating acid stress in E. coli and Salmonella typhimurium (Chakraborty and Kenney, 2018, Chakraborty et al., 2015, Quinn et al., 2014). OmpR also increases the adhesion properties of E. coli cells by controlling the expression of curli (Perni et al., 2016). Many new novel targets are also regulated by OmpR in E. coli (Shimada et al., 2015). In addition, OmpR is directly or indirectly associated with the regulation of virulence genes in pathogens and so is highly responsible for pathogenesis (Bernardini et al., 1990, Brzostek et al., 2003, Dorman et al., 1989, Feng et al., 2003, Lee et al., 2000, Lin et al., 2018, Ogasawara et al., 2007, Pickard et al., 1994, Vidal et al., 1998). Sequence alignment of OmpR from different bacterial pathogenic species shows that the protein is conserved (Figure S1), so OmpR is a global regulator. Despite growing information about the importance of OmpR, the molecular and structural bases of the mechanisms that drive OmpR to regulate a large number of genes remain elusive.

OmpR belongs to the OmpR/PhoB family of RRs, which is characterized by the distinctive winged helix-turn-helix structure of its DNA-binding domain (DBD) (Kondo et al., 1997, MartinezHackert and Stock, 1997). In RRs, this motif mainly includes a transactivation loop and β-hairpin structure that are responsible for interacting with and binding to the target DNA (Brennan, 1993, Lai et al., 1993). Full-length OmpR (OmpR-FL) has two domains: an N-terminal receiver domain (OmpRn) and a C-terminal DNA-binding domain (OmpRc). In traditional activation mechanism of RRs, phosphorylation of the Asp residue in the N-terminal receiver domain activates the protein. This activation is believed to induce the conformational changes that promote dimerization of the protein for binding to the target DNA from its C-terminal DNA-binding domain and modulate gene expression for an adaptive response (Gao et al., 2007, Jeon et al., 2001, Toro-Roman et al., 2005). However, in OmpR, either OmpR–DNA interactions enhance phosphorylation (Ames et al., 1999), or phosphorylation may enhance OmpR–DNA interactions (Aiba et al., 1989, Head et al., 1998). Also, OmpR dimerization is suggested to be required for DNA binding (Harlocker et al., 1995). Therefore, the activation mechanism could be controlled by many other factors and the exact reaction steps may vary for different responses (Barbieri et al., 2013, Rhee et al., 2008, Yoshida et al., 2006). Another feature of OmpR/PhoB family proteins is a short linker that connects the N-terminal receiver domain and C-terminal DNA-binding domain. The linker region of OmpR is 15 amino acid residues long, longer than those for other OmpR/PhoB family proteins (Figure S2). The major role of the linker region is unknown; however, this difference in linker length may have an important role in the inter- and intra-domain interactions of OmpR (Mattison et al., 2002, Walthers et al., 2003).

Many RR protein structures in the OmpR/PhoB family have been determined. Five full-length structures in the apo-form [DrrD (Buckler et al., 2002), DrrB (Robinson et al., 2003), RegX3 (King-Scott et al., 2007), MtrA (Friedland et al., 2007) and PrrA (Nowak et al., 2006)] and two full-length in complex with the promoter DNA structures [KdpE (Narayanan et al., 2014) and PmrA (Lou et al., 2015)] have been reported. Structural information about these related proteins highlights the interface between the N-terminal and C-terminal domains and reveals the DNA binding mechanism of RRs. However, sequence alignment of these OmpR/PhoB family proteins indicated that the interacting residues are not fully conserved (Figure S2). For OmpR, crystal and solution structures of only the OmpRc in the apo-form have been reported (Kondo et al., 1997, MartinezHackert and Stock, 1997, Rhee et al., 2008). The structure of OmpR-FL or OmpR-FL in complex with promoter DNA is still unknown. Also, phosphorylated OmpR-FL has low solubility in solution (Barbieri et al., 2013). Therefore, because of the dynamic nature of OmpR, structural investigation of phosphorylated OmpR-FL and OmpR-FL in complex with its DNA is difficult. In a previous NMR study, DNA contact residues of OmpRc critical to the C1 region of the ompC promoter DNA interaction were identified (Rhee et al., 2008). In addition, OmpR may bind to its promoter DNA in head-to-head or head-to-tail dimer orientation (Maris et al., 2005, Rhee et al., 2008).

In this study, we report the first crystal structure of E. coli OmpRc in complex with the F1 region of the ompF promoter DNA. The structure showed that a compact asymmetrical dimer of OmpRc binds at two adjacent sites on F1-DNA. The dimer interface indicates that the positively charged surface of OmpRc matches the phosphate backbone of DNA. As characterised in other OmpR/PhoB family RRs, specific contacts were observed from the OmpRc recognition helix with the DNA major groove containing GT bases and the wing region with the minor groove. Biophysical experiments such as nuclear magnetic resonance (NMR), fluorescent polarization and thermal stability analysis using circular dichroism spectroscopy confirmed previous observations that OmpR-FL can interact with the ompF and ompC promoter DNA sequence without phosphorylation. Furthermore, we determined a unique domain-swapped dimeric form of OmpRc under different crystallization conditions. This structural study reveals the DNA binding mechanism in the OmpR/PhoB family RR. Moreover, it can help with inhibitor screening for this potential “druggable” target.