Structural Characterization of Brucella Lov-Histidine Kinase (Comunicación Oral)

JIMENA RINALDI; SEBASTIÁN KLINKE; MARIANA GALLO; GABRIELA SYCZ; MARTIN ARAN; GASTÓN PARIS; DANIEL O. CICERO; FERNANDO A. GOLDBAUM

Córdoba

Congreso; 16th International Congress on Photobiology; 2014

International Union of Photobiology

Light modulates the virulence of the bacterium Brucella through a histidine kinase (HK) containing a light-oxygen-voltage (LOV) domain [1]. In order to understand how light modulates the kinase activity we started a structural characterization of the molecule in its light and dark conformations. Crystal structure of the Brucella LOV domain shows it adopts the typical alpha/beta PAS domain fold, consisting of a beta-sheet and alpha-helical connector elements. NMR studies point to the beta-scaffold as a key element in the light activation [2]. According to secondary structure predictions, Brucella LOV-HK harbors a C-terminal helix (J-helix) contiguous to the LOV core, which is estimated to be 37 residues long and a 25 residue long N-terminal helix (N-helix), with no sequence similarity to other known LOV proteins. Solution experiments show that the N-helix is essential for the formation of a stable dimer and that, under the experimental conditions, the J-alpha helix is 17 residue-long, flexible and exposed to solvent. The N-terminus of the J-helix changes its chemical environment upon illumination suggesting a pivotal role. On the other hand, we solved the crystal structure of the HK domain. This is the first structure of a histidine kinase from the bioinformatically identified HWE family [3]. It presents a Dimerization and Histidine phosphotransfer subdomain and a Catalytic and ATP-binding subdomain. The structure showstwo different dimer conformations. SLS experiments indicate the HK domain is a monomer in solution. These observations suggest that the HK domain alternates between the two dimeric conformations. Mutational analysis confirms the existence in solution of the two dimeric and the monomeric conformations. The function of these forms will be our next focus.[1] Swartz, T.E. et al. (2007). Science 317, 1090-1093.[2] Rinaldi, J. et al. (2012). J. Mol. Biol. 420, 112-127.[3] B. Karniol & R. Vierstra. (2004) J. Bact., 186, 445-453.