Structural modeling of the ExuR and UxuR transcription factors ofE. coli: search for the ligands affecting their regulatory properties

Abstract

Gammaproteobacteria get energy for their growth from different carbon sources using either glycolysis or alternative metabolic pathways induced in stress conditions. These metabolic switches are coordinated by complex interplay of regulatory proteins sensing concentrations of available metabolites by mechanisms yet to be understood. Here, we use two transcriptional regulators, ExuR and UxuR, controlling d-galacturonate (d-gal) and d-glucuronate metabolism in Escherichia coli, as the targets for computational search of low-molecular compounds capable to bind their ligand-binding domains. Using a flexible molecular docking, we modeled the interactions of these proteins with substrates and intermediates of glycolysis, Ashwell and Entner-Doudoroff pathways. For UxuR, the two preferred sites of ligand binding were found: one is located within the C-terminal domain, while another occupies the interdomain space. For ExuR, the only one preferred site was detected in the interdomain area. Availability of this area to different ligands suggests that, similar to the Lac repressor, the DNA-binding properties of UxuR and ExuR may be changed by repositioning of their domains. Experimental assays confirmed the ability of ligands with highest affinities to bind the regulatory proteins and affect their interaction with DNA. d-gal that is carried into the cell by the ExuT transporter appeared to be the best ligand for repressor of the exuT transcription, ExuR. For UxuR, the highest affinity was found for d-fructuronate transported by GntP, which biosynthesis is repressed by UxuR. Providing a feedback loop to balance the concentrations of different nutrients, such ligand-mediated modulation can also coordinate switching between different metabolic pathways in bacteria.

Control of hexuronate metabolism in Escherichia coli by the two interdependent regulators, ExuR and UxuR: derepression by heterodimer formation

“…Although the amount of the binary complex was unchanged, the amount of unbound DNA decreased and was replaced by stuck complexes at the top of the gel. This might be due to glucuronate-induced UxuR oligomerization (Tutukina et al, 2016). The effect of an exuR deletion on the levels of uxuR transcription was determined by qPCR (Fig.…”

Molecular insights into effector binding by DgoR, a GntR/FadR family transcriptional repressor of D‐galactonate metabolism in Escherichia coli

“…These TRs repress sugar acid metabolic genes, and their negative regulation is relieved upon binding of cognate effector, the substrate itself (B. subtilis GntR: D-gluconate and E. coli DgoR: D-galactonate), a metabolic intermediate (Polaromonas sp. strain JS666 GguR: 5-keto-4-deoxy-D-glucarate/galactarate) or both (E. coli ExuR: D-galacturonate and D-glucuronate and E. coli UxuR: D-glucuronate and D-fructuronate) (Bates Utz et al, 2004;Bouvier et al, 2019;Fujita and Miwa, 1989;Miwa and Fujita, 1988;Singh et al, 2019;Tutukina et al, 2016a;2016b;Yoshida et al, 1993).…”

“…Regarding the molecular details of effector-regulator interactions, the amino acids involved in effector binding have been examined for GntR; however, only two residues were identified (Yoshida et al, 1995). Further, although molecular docking efforts have been made to predict the effector-binding pocket in ExuR and UxuR (Purtov et al, 2019;Tutukina et al, 2016b), there is no experimental validation of the site. Investigation of the effector-regulator interactions is, therefore, required for a molecular understanding of sugar acid-responsive regulation in the FadR subfamily.…”

Transcription factor allosteric regulation through substrate coordination to zinc

“…−35 and −10 sequences and transcription start site are capitalized, with the binding site of UxuR located between the −35 and −10 sequences (Aaattggtataccaattt). −35 and −10 sites are from promoter J23101 from the Registry of Standard Biological Parts ( http://www.partsregistry.org ) and the UxuR binding site is from the promoter of the uxuR gene from E. coli ( 20 ). This PCR product and the pSNR1 backbone were both digested with SacI and AflII and ligated together.…”

“…However, the conformational changes that lead to this transcriptional regulation are poorly understood. Previous modeling efforts for UxuR TF assumed that the structure was a monomer or did not consider the hypothesis of metal binding ( 20 , 21 ). Consequently, until now, the ligand-binding site and the residues involved remain unknown, as well as the associated conformational changes.…”