Crystallographic structure of the PhyR response regulator from the pathogenic bacterium Brucella abortus

SEBASTIÁN KLINKE; GABRIELA SYCZ; JIMENA RINALDI; GASTÓN PARIS; FERNANDO A. GOLDBAUM

Santa Fe

Congreso; VIII Reunión Anual de la Asociación Argentina de Cristalografía; 2012

Asociación Argentina de Cristalografía

Two-component signal transduction systems are modules that allow bacteria to rapidly adapt to changing environmental conditions. In the most common case, they are formed by a sensor histidine kinase (HK) which, upon sensing of an external signal, is able to phosphorylate a cognate response regulator (RR). The latter protein undergoes structural changes that are able to modify gene expression by directly binding to DNA, catalyze metabolic reactions or alter protein-protein interactions. The pathogenic bacterium Brucella abortus, the causative agent of brucellosis, bears a particular two-component system formed by a blue-light sensor HK called LOV-HK and a RR called PhyR. The activation of LOV-HK has been shown to increase the virulence of this pathogen. Additionally, a mutant in the LOV-HK gene presents reduced infectivity, suggesting that this system is a virulence factor and an important regulatory system during the infection process. As part of our project, we aimed to solve the three-dimensional structure of PhyR. We crystallized PhyR using a mixture containing 22% PEG 4000, 11% isopropanol and 0.1 M sodium acetate. Diffraction data were then collected at 2.05 Å resolution and the structure was solved by the molecular replacement method using the homologous PhyR protein from the bacterium Caulobacter crescentus as search model. A single molecule was found in the asymmetric unit in the tetragonal spacegroup P43212 with cell parameters a = b = 53.37 Å, c = 177.30 Å. The final model comprises 268 residues, 1 calcium cation as ligand and 45 water molecules, with R = 0.212 and Rfree = 0.247. Figure 1 shows the overall structure of the protein. It is composed by two separated domains: a receiver domain at the C-terminus, which is phosphorylated by LOV-HK, and an effector domain at the N-terminus, which is proposed to bind a specific anti-sigma factor called NepR, activating the general stress response in Brucella. In our poster, we will present a detailed description of PhyR and a comparison with the previously known structures of this protein in C. crescentus and Sphingomonas sp. Fr1. Additionally, we will discuss ideas for the future structural characterization of the LOV-HK - PhyR complex. This work was supported by CONICET and ANPCyT. We acknowledge access to the PROXIMA 1 beamline at the SOLEIL Synchrotron (France).