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

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

San Luis

Congreso; XII Reunión Annual de la Asociación Argentina de Cristalografía; 2016

Asociación Argentina de Cristalografía

Light modulates the virulence of the bacterium Brucella abortus through a histidine kinase containing a light-oxygen-voltage domain sensitive to blue light (LOV-HK) [1, 2]. This photoreceptor is involved in the general stress response system [3]. In order to understand how light modulates the kinase activity we started a structural characterization of the molecule in its light and dark conformations. The LOV domain consists of a globular core and N- and C-terminal flanking regions. Its crystal structure of the Brucella LOV domain shows that the core adopts the typical α/β PAS domain fold, consisting of a β-sheet and α-helical connector elements. Also, the LOV domain structure reveals an N-terminal α-helix that plays a central role in dimerization, a finding that was further confirmed by light scattering experiments. NMR studies point to the β-scaffold as a key element in the light activation, as well as the N-terminus of the J-helix (C-terminal) whose chemical environment changes upon illumination, suggesting a pivotal role [4]. We also studied the dark recovery kinetics of the LOV domain by UV-Vis spectroscopy, and comparing the results from different constructs it can be deduced that the presence of the N-helix increases dramatically the half-life of the lit state.On the other hand, we solved the crystal structure of the histidine kinase domain (HK) [5]. It presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical histidine kinase parallel homodimers (C), and an antiparallel non-canonical (NC) dimer[6]. Using cross-linking experiments, we showed that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK.