Many proteobacteria are able to monitor their population densities through the release of pheromones known as N-acylhomoserine lactones. At high population densities, these pheromones elicit diverse responses that include bioluminescence, biofilm formation, production of antimicrobials, DNA exchange, pathogenesis and symbiosis. Many of these regulatory systems require a pheromone-dependent transcription factor similar to the LuxR protein of Vibrio fischeri. Here we present the structure of a LuxR-type protein. TraR of Agrobacterium tumefaciens was solved at 1.66 A as a complex with the pheromone N-3-oxooctanoyl-L-homoserine lactone (OOHL) and its TraR DNA-binding site. The amino-terminal domain of TraR is an alpha/beta/alpha sandwich that binds OOHL, whereas the carboxy-terminal domain contains a helix turn helix DNA-binding motif. The TraR dimer displays a two-fold symmetry axis in each domain; however, these two axes of symmetry are at an approximately 90 degree angle, resulting in a pronounced overall asymmetry of the complex. The pheromone lies fully embedded within the protein with virtually no solvent contact, and makes numerous hydrophobic contacts with the protein as well as four hydrogen bonds: three direct and one water-mediated.

Some of the Escherichia coli cells produce plasmid-encoded toxic colicins that are secreted to kill closely related bacteria cells thereby they have better survival advantage during times of stress. The colicin E7 (ColE7) is one of the nuclease-type colicins, which contains a non-specific nuclease domain capable of hydrolyzing DNA in target cells. The nuclease activity of ColE7 is inhibited by the co-expressed immunity E7 protein (Im7), which binds to the nuclease domain of ColE7 in the host cell. The nuclease domain of ColE7 (nucleaseColE7) contains a HNH motif, which has been identified in many endonucleases. The crystal structure of the phosphate-bound nucleaseColE7/Im7 complex determined at 2.0 Å resolution with different metal ions showed that a phosphate ion was bound directly to the zinc ion in the HNH motif, suggesting that the zinc ion not only stabilizes the folding of the enzyme, but is also likely involved in DNA hydrolysis. Several residues located at positions close to the zinc-binding site were mutated to alanine and these mutants showed decreased or no nuclease activities. A gel retardation assay further demonstrated that the nuclease-ColE7 hydrolyzed DNA in the presence of zinc ions, but only bound to DNA in the absence of zinc ions. These results demonstrate that the zinc ion in the HNH motif of nucleaseColE7 is not required for DNA binding, but is essential for DNA hydrolysis, suggesting that the zinc ion not only stabilizes the folding of the enzyme, but is also likely involved in DNA hydrolysis. Many proteobacteria release N-acylhomoserine lactones as pheromones to measure their population density and to initiate gene expression at high cell density, resulting in diverse responses, including bioluminesence, biofilm formation, production of antimicrobials, DNA exchange, pathogenesis, and symbiosis (Whitehead et al., 2001). Many of these regulatory systems require a pheromone-dependent transcription factor similar to LuxR of Vibrio fisheri, whose N-terminal domain binds the pheromone and whose C-terminal domain binds DNA. Here we present the structure of the LuxR-type protein TraR protein of Agrobacterium tumefaciens complexed with the pheromone N-3-oxooctanoyl-L-homoserine lactone (OOHL) and a DNA at a resolution of 1.6 Å. The amino terminal domain of TraR (169 amino acids) is an α β α sandwich that binds OOHL, while the carboxyl terminal domain (60 amino acids) contains a helix-turn-helix motif DNA binding domain. The TraR dimer displays a two-fold rotational symmetry in each domain. However, these two axes of symmetry intersect at a 90° angle resulting in a pronounced overall asymmetry. The pheromone lies fully engulfed within the protein with virtually no contact to bulk solvent, and is stabilized by numerous hydrophobic interactions and by three direct hydrogen bonds and one water-mediated hydrogen bond. Keywords: COLICIN, DNASE, HNH MOTIF d(T[c,s]T)) and tetranucleotide (d(GT[c,s]TG). In these complexes, the sidechains of Tyr 32L, His 34L, Arg 50L, Ser 95H and Pro 100H, and the Asn 31L ...