Tetrameric architecture of an active phenol-bound form of the AAA+ transcriptional regulator DmpR

Kwang-Hyun Park,Sungchul Kim,Eui-Jeon Woo,Arti Baban Dumbrepatil,Hyung-Nam Song,Victoria Shingler,Seung-Goo Lee,Vinod Vikas Patil,Jee-Eun Cho,Su-Jin Lee,Chirlmin Joo,Woo-Chan Ahn

doi:10.1038/s41467-020-16562-5

Abstract

The Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein (bEBP) from the AAA+ ATPase family. Even though it was discovered more than two decades ago and has been widely used for aromatic hydrocarbon sensing, the activation mechanism of DmpR has remained elusive. Here, we show that phenol-bound DmpR forms a tetramer composed of two head-to-head dimers in a head-to-tail arrangement. The DmpR-phenol complex exhibits altered conformations within the C-termini of the sensory domains and shows an asymmetric orientation and angle in its coiled-coil linkers. The structural changes within the phenol binding sites and the downstream ATPase domains suggest that the effector binding signal is propagated through the coiled-coil helixes. The tetrameric DmpR-phenol complex interacts with the σ54 subunit of RNA polymerase in presence of an ATP analogue, indicating that DmpR-like bEBPs tetramers utilize a mechanistic mode distinct from that of hexameric AAA+ ATPases to activate σ54-dependent transcription.

Highlights

The Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein from the AAA+ ATPase family
As is typical of bacterial enhancer binding protein (bEBP), DmpR consists of three domains: [1] a sensory domain consisting of a vinyl-4-reductase (V4R) scaffold that functions in binding of an aromatic effector molecule11–13, [2] a conserved central AAA+ ATPase domain bearing the bEBPspecific GAFTGA motif that is involved in coupling ATP hydrolysis to the restructuring of σ54-RNAP, and [3] a DNA
Many AAA+ ATPases function as hexamers, the active oligomeric state of DmpR-like bEBPs remained unclear

Summary

Introduction

The Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein (bEBP) from the AAA+ ATPase family. Even though it was discovered more than two decades ago and has been widely used for aromatic hydrocarbon sensing, the activation mechanism of DmpR has remained elusive. As is typical of bEBPs, DmpR consists of three domains: [1] a sensory domain consisting of a vinyl-4-reductase (V4R) scaffold that functions in binding of an aromatic effector molecule11–13, [2] a conserved central AAA+ ATPase domain bearing the bEBPspecific GAFTGA motif that is involved in coupling ATP hydrolysis to the restructuring of σ54-RNAP, and [3] a DNA binding domain that interacts with the palindromic upstream activating sites (UASs) situated ~100–200 bp upstream from the σ54 promoter. DmpR share high sequence homology with other aromatic-responsive bEBPs, such as XylR, TouR, PoxR and MopR, and this subgroup are known to transition from inactive dimers to active oligomers upon the binding of an aromatic effector compound as a prerequisite for their capacity to direct σ54-dependent transcription

Methods

Results

Conclusion