KLINKE Sebastian
congresos y reuniones científicas
Structural studies on the protein LOV-Histidine kinase from Brucella abortus
Congreso; VII Reunión Anual de la Asociación Argentina de Cristalografía; 2011
Institución organizadora:
Asociación Argentina de Cristalografía
LOV (Light, Oxygen, Voltage) sensory domains are found in Archaea, Eukarya and Bacteria. In the latter, most of the LOV domain-containing proteins are associated with histidine kinase (HK) output domains involved in signal transduction. The HK protein is the first component of two-component systems, which are involved in environmental sensing in bacteria regulating gene expression, chemotaxis and virulence. LOV domains bind FMN as cofactor and undergo a photocycle upon illumination. Blue light excites the FMN molecule to the triplet state, giving rise to a covalent adduct between the ligand and a cysteine residue from the protein. In the darkness, the covalent adduct decays thermally to the basal state. Recent evidence suggests that the cysteinyl adduct is the signal state of prokaryotic LOV-HKs. Bacteria from the genus Brucella are intracellular pathogens that cause a worldwide zoonosis named brucellosis. A protein containing a LOV domain followed by a PAS and an HK domain has been recently identified in Brucella abortus. This protein binds FMN and undergoes a photocycle but it does not decay upon incubation in the darkness. Infection of macrophage cells with a Brucella mutant lacking the LOV gene shows an attenuated phenotype suggesting that the LOV-HK protein is a virulence factor [1]. Macrophage infection with Brucella wild type cells grown in the dark also shows a decrease in the number of intracellular bacteria when compared with wild type cells grown in light, confirming the effect of light in bacterial virulence. As part of this project we aim to solve the crystallographic structure of LOV-HK, which will allow us a better understanding of the signal transduction effect between the LOV and HK domains, giving clues about the light sensing mechanism of LOV proteins and the general activation of histidine kinases. Additionally, we aim to explain the virulence enhancement by light in Brucella. In this talk we will show our recent progress in the project, describing the LOV domain structure at 1.64 Å resolution and preliminary results on HK domain crystals. In addition, we will also illustrate the different strategies that are being followed at the moment to cover as much as possible of the whole LOV-HK protein in single constructs.   [1] Swartz, T.E. et al. (2007). Science317, 1090-1093.