Structural basis of a blue light activated signaling pathway involved in Brucella virulence

RINALDI, J.; GALLO, M.; ARAN, M.; PARIS, G.; MARTÍ, M.A.; FERNÁNDEZ, I.; SCYCZ, G.; KLINKE S.; BONOMI, H.R.; ARRAR, M.; ESTRÍN, D.A.; CERUTTI, M.L; CICERO, D.O.; GOLDBAUM, F.A

San Luis

Congreso; XII Reunión Anual de la Sociedad Argentina de Cristalografía; 2016

Asociación Argentina de Cristalografía

Light modulates the virulence of the bacterium Brucella abortus through a histidinekinase containing a light-oxygen-voltage domain sensitive to blue light (LOV-HK) [1, 2]. This photoreceptor is involvedin the general stress response system [3].In order to understand how light modulates the kinase activity we started astructural characterization of the molecule in its light and darkconformations. The LOV domain consists of a globular core and N- and C-terminalflanking regions. Its crystalstructure of the Brucella LOV domainshows that the core adopts the typical α/β PAS domain fold, consisting of aβ-sheet and α-helical connector elements. Also, the LOV domain structurereveals an N-terminal α-helix that plays a central role in dimerization, afinding that was further confirmed by light scattering experiments. NMR studiespoint to the β-scaffold as a key element in the light activation, as well asthe N-terminus of the J-helix (C-terminal) whose chemical environment changesupon illumination, suggesting a pivotal role [4]. We also studied the dark recovery kinetics of the LOV domain byUV-Vis spectroscopy, and comparing the results fromdifferent constructs it can be deduced that the presence of the N-helixincreases dramatically the half-life of the lit state.On the other hand, we solved the crystalstructure of the histidine kinase domain (HK) [5]. It presents two different dimeric assemblies in the asymmetricunit: one similar to the already described canonical histidine kinase parallelhomodimers (C), and an antiparallel non-canonical (NC) dimer[6]. Using cross-linking experiments, we showed that the Cdimer is the functionally relevant species. Mutational analysis demonstratesthat the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed forthe NC and C states highlight the large conformational flexibility of the HKdomain. Through the analysis of these alternative conformations by means ofmolecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK. [1] T.E. Swartz, et al. (2007)Science 317, 1090-1093.[2] C.R. Gourley et al. (2014)Pathogen. Dis. 15, 1-8.[3] G. Sycz et al. (2015) PLoSOne 10,  e0124058. [4] J. Rinaldi et al. (2012) J.Mol. Biol.  420, 112-127. [5] S. Klinke et al. (2015) ActaCrystallogr. Sect. D: Biol. Crystallogr. 71, 1433-43. [6] J. Rinaldi et al. (2016) J.Mol. Biol.  428, 1165-79.