Structural basis of a blue light activated signaling pathway involved in Brucella virulence (Conferencia Semiplenaria)

JIMENA RINALDI; SEBASTIÁN KLINKE; IGNACIO FERNÁNDEZ; MARIANA GALLO; MEHRNOOSH ARRAR; GABRIELA SYCZ; MARTIN ARAN; MARÍA L. CERUTTI; HERNÁN R. BONOMI; GASTÓN PARIS; DANIEL O. CICERO; DARÍO A. ESTRÍN; MARCELO A. MARTÍ; FERNANDO A. GOLDBAUM

San Luis

Congreso; XII Reunión Anual de la Asociación Argentina de Cristalografía (AACr); 2016

Asociación Argentina de Cristalografía (AACr)

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 alpha/beta PAS domain fold, consisting of a beta-sheet and alpha-helical connector elements. Also, the LOV domain structurereveals an N-terminal alpha-helix that plays a central role in dimerization, afinding that was further confirmed by light scattering experiments. NMR studiespoint to the beta-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 crystal structureof the histidine kinase domain (HK) [5].It presents two different dimeric assemblies in the asymmetric unit: onesimilar 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 theC dimer 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.