RESUMO
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.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Conformação Proteica , Multimerização Proteica , Transativadores/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Fenol/metabolismo , Ligação Proteica , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Homologia de Sequência de Aminoácidos , Transativadores/genética , Transativadores/metabolismoRESUMO
Repair of uracils in DNA is initiated by uracil DNA glycosylases (UDGs). Family 1 UDGs (Ung) are the most efficient and ubiquitous proteins having an exquisite specificity for uracils in DNA. Ung are characterized by motifs A (GQDPY) and B (HPSPLS) sequences. We report a novel dimeric UDG, Blr0248 (BdiUng) from Bradyrhizobium diazoefficiens. Although BdiUng contains the motif A (GQDPA), it has low sequence identity to known UDGs. BdiUng prefers single stranded DNA and excises uracil, 5-hydroxymethyl-uracil or xanthine from it. BdiUng is impervious to inhibition by AP DNA, and Ugi protein that specifically inhibits family 1 UDGs. Crystal structure of BdiUng shows similarity with the family 4 UDGs in its overall fold but with family 1 UDGs in key active site residues. However, instead of a classical motif B, BdiUng has a uniquely extended protrusion explaining the lack of Ugi inhibition. Structural and mutational analyses of BdiUng have revealed the basis for the accommodation of diverse substrates into its substrate binding pocket. Phylogenetically, BdiUng belongs to a new UDG family. Bradyrhizobium diazoefficiens presents a unique scenario where the presence of at least four families of UDGs may compensate for the absence of an efficient family 1 homologue.
Assuntos
Proteínas de Bactérias/metabolismo , Bradyrhizobium/enzimologia , Reparo do DNA , DNA Bacteriano/metabolismo , Uracila-DNA Glicosidase/metabolismo , Uracila/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Bradyrhizobium/genética , Clonagem Molecular , Cristalografia por Raios X , Dano ao DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Uracila-DNA Glicosidase/química , Uracila-DNA Glicosidase/genéticaRESUMO
Positive phenol-degradative gene regulator (PoxR) is a σ(54)-dependent AAA+ ATPase transcription activator that regulates the catabolism of phenols. The PoxR sensory domain detects phenols and relays signals for the activation of transcription. Here we report the first structure of the phenol sensory domain bound to phenol and five derivatives. It exists as a tightly intertwined homodimer with a phenol-binding pocket buried inside, placing two C termini on the same side of the dimer. His102 and Trp130 interact with the hydroxyl group of the phenol 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. A shift at the C-terminal helix suggests that there is a possible conformational change upon ligand binding. The results provide a structural basis of chemical effector binding for transcriptional regulation with broad implications for protein engineering.
