Elucidating how bacteria sense phenols through structural analysis of transcription activators

다국어 초록 (Multilingual Abstract)

Some bacteria are well equipped to utilize phenol as a carbon and energy source. Positive phenol-degradative gene regulators such as PoxR from Ralstonia eutropha and DmpR (CapR) from Pseudomonas putida are σ54-dependent AAA+ ATPase transcription activators that regulate the catabolism of phenols. The PoxR sensory domain detect phenols and relays signals for the activation of transcription. My thesis deals with understanding the structure and function of phenol responsive transcription activators. Based on function, these transcription activators can be divided into three domains viz. sensory domain, ATPase domain and DNA binding domain.
The analysis of sensory domain revealed that it exists as a tightly intertwined homodimer with a ligand binding pocket buried inside, placing two Carboxyl termini on the same side of the dimer. Whereas the whole protein exists as a tetramer upon binding of phenol. H102 and W130 in case of PoxR and H100 and W128 in case of DmpR interact with the hydroxyl group of the phenols in a cavity surrounded by rigid hydrophobic residues on one side and a flexible region on the other. Each monomer has a V4R fold with a unique zinc-binding site. Upon binding of ligand, a shift at the Carboxyl terminal helix suggests that there is a possible conformational change.
The linker region that connects sensory domain to central ATPase domain is helical. The first half of linker region (~225 residues) is quite rigid made up of helix that run parallel making slanting angles in a dimer. The latter half of linker is quite flexible having higher B-factor.
The structural analysis shows that these residues are very critical for nucleotide binding. The binding of phenol produces shifts in the linker region causing Y233 and Q232 to make space for accommodation of nucleotide. The pi-pi stacking interaction of aromatic ring of tyrosine in nucleotide helps in binding of nucleotide in cleft. The phenol unbound form would not make any space for stable binding of nucleotide thus lowering overall ATPase activity and oligomerization. In absence of phenol the DmpR is in dynamic state of dimer and tetramer. The addition of phenol promotes the tetramer formation from dimers. The sensory domain interacts with ATPase domain from another dimer thus giving it a stability. The arrangement of ATPase domain in a tetramer is different from typical NTRC family proteins although the ATP binding cavity and key residues are quite similar. The AAA+ domains have the characteristic Walker A and Walker B motifs. The GAFTGA motif is located at the α/β subdomain surface at the tip of loop. ATPase domain exists as a monomer independently. The ATPase domain physically interacts with sensory domain. The GAFTGA (310-315) loop of ATPase domain interacts with the VNTLGI (53-58) R50 and D140 of sensory domain. The orientation of ATPase domain is different from the arrangement of reported ligand bound ATPases from NTRC family. There are other important residues of ATPase domain that have interaction with sensory domain (Sensory domain: N54, IL58, E59, D140, N145, D146, D194, 195, S198, N201, Y202, K204, and D206. ATPase domain: D298, A311, F312, P341, R342, A343, S346, R349, G360, N362, T364, R389). Sensory domain is critical for ATPase activity. Elimination of DNA binding domain adversely affects the ATPase activity. This thesis work is the first report about the structural arrangement of phenol-responsive transcription activator that can provide a structural basis of chemical effector binding for transcriptional regulation with broad implications for protein engineering, strain improvement and biosensor development.