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

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.

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Eui-jeon Woo / Chirlmin Joo Apr 29, 2020 1. Protein crystal diffraction data were collected from Pohang Accelerator Laboratory 'MX7A beamline'. The collected diffraction data was processed using 'HKL2000'. 2. Single-molecule data was collected using a modified version of "SINGLE" software that was developed by the lab of Prof. Dr. Ha. The original version is available at: https://cplc.illinois.edu/software/.
1. 'CCP4 5.1', 'Phenix 1.14' and 'wincoot 0.8.9' were used for protein structure determination and analysis. 2. Molecular images were produced using 'Pymol 2.0'. 3. The dimer structure was made using 'Discovery Studios 2.0' 4. MALS data were collected and analysed using 'ASTRA 6' 5. ITC data were analysed with the MicroCal 'Origin 5.0' software package 6. Single-molecule data were first analyzed using custom IDL scripts that allowed automatic extraction of single-molecule time trajectories. Single-molecule trajectories were analyzed using custom MATLAB scripts that allowed manual selection and processing of the trajectories. Statistical analysis was performed using both standard functions in IDL (version 8.2), MATLAB R2017b, OriginPro 2015 (version Sr1 b9.2.257) and Microsoft Excel365. Figure panels for Fig. 1, Fig. 6 and ED Fig. 8  Experiments that yielded a single value per data point were performed as three independent experimental replicates.
For the single-molecule photobleaching assays, all traces were selected and analyzed that are exhibited by sufficient amount of intensity intially and followed by step-wise decreasing until a basal level intensity. In contrast, traces that displayed compicated (e.g. fluctuation, nothing end at the trajectory) were excluded from the analysis as these molecules may correspond to false positive signals. In some cases attempts to reproduce data on microscope slides of low quality (poor surface-passivation) failed. These datasets were excluded and repeated on high-quality microscope slides.
To verify the reproducibility, three-five individual experiments were performed and analyzed. We hereby confirm we can reproduce the data presented in this manuscript. All presented data is representative of three-five replicates that yielded similar results.
To allow accurate comparison between the data presented in the figures, the substrates that were used for each figure were grouped and the data was obtained at the same day. Randomization is not applicable in this case.
Analysis performed in this manuscript were not blinded. However, strict selection criteria were used to ensure reproducibility of the analysis.