Characterisation of the Putative Effector Interaction Site of the Regulatory HbpR Protein from Pseudomonas azelaica by Site-Directed Mutagenesis

Abstract

Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ54-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate PC promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.

An Effective Strategy for a Whole-Cell Biosensor Based on Putative Effector Interaction Site of the Regulatory DmpR Protein

“…Mutagenesis was performed by using the mutagenic PCR [26] . The primers used for site-directed mutagenesis are as follows, (a) for F42L (to generate pRLuc42R) forward primer: TGTTGCTGCAG CTT TCAGCGATGG and reverse primer: GCCATCGCTGA AAG CTGCAGCAA (b) for D140E and Q143L (to generate pRLuc140p143R) forward Primer: ACC GAA CTGGGG CTG ATGCA and reverse primer: TGCAT CAG CCCCA GTT CGGT (c) for L113M and D116A (to generate pRLuc113p116R) forward Primer: ACCGAG ATG GATATC GCC AAGGAA and reverse primer: TTCCTT GGC GATATC CAT CTCGGT (Altered codons are underlined).…”

Towards the Response Threshold for p -Hydroxyacetophenone in the Denitrifying Bacterium “Aromatoleum aromaticum” EbN1

“…As typical for prokaryotes in general (26,27), these transcriptional regulators are mostly of the one-component type. Major research focused on deciphering promoter interactions (28,29), architecture of effector-specific transcriptional networks (30), and the structural basis of effector binding to the regulator (31)(32)(33)(34). Mechano-transcriptional activators of the NtrC family bind upstream of the promoter, stimulating open complex formation of the 54 -RNA-polymerase holoenzyme (35,36).…”

Transcriptional regulatory proteins as biosensing tools

“…The regulatory proteins ArsR, HbpR-A family of proteins, and cyclic AMP receptor protein (CRP) have been shown to undergo conformational changes upon binding their respective ligands. 25–28…”

“…To explore potential use of other regulatory proteins in biosensing, we chose HbpR, a protein known to bind hydroxylated polychlorinated biphenyls, OH-PCBs. 28,33 HbpR has four distinct domains. The A-domain interacts directly with an effector molecule causing a drastic conformational change relayed to the rest of the protein through a helix linker region in the B-domain, which is translated to the C-domain, whose ATPase activity is then triggered recruiting σ54 RNA polymerase.…”

“…34 This class of proteins are difficult to express, purify, and stabilize in solution, and, as a result, there is sparse structural information. 28,35 Homology studies have been performed, 28,35 but still there is a lack of specific information on the overall protein structure. This makes our prior rational design strategy of these protein biosensors a nearly impossible proposition.…”