CONICET | Buscador de Institutos y Recursos Humanos

Lightmodulates the virulence of the bacterium Brucella abortus through a histidinekinase containing a light-oxygen-voltage domain sensitive to blue light(LOV-HK). Brucella LOV-HK exhibits the spectroscopic changes corresponding tothe typical adduct formation of LOV domains, between the C4 of the isoalloxazinering and S of a strictly conserved cysteine. After illumination, BrucellaLOV-HK does not return to the dark state. Brucella LOV-HK increases itsautophosphorylation upon absorption of blue light. Brucella LOV-HK comprises anN-terminal blue-light sensor (LOV) domain, followed by a central PAS domain anda C-terminal histidine kinase (HK) domain. We have performed a structuralcharacterization of the isolated LOV and HK domains. The LOV domain consists ofa globular core and N- and C-terminal flanking regions. The core of the LOVdomain adopts the typical alpha/beta PAS domain fold, consisting of a beta-sheetand alpha-helical connector elements. The beta-scaffold is a central element inthe light activation. The N-terminal region consists of an alpha-helix andplays an essential role in dimerization. The HK is a parallel dimer. It iscomprised of the dimerization/histidine phosphoacceptor subdomain (DHp) and thecatalytic and ATP-binding subdomain (CA), which are connected by a flexible linker.Based on the information from the study of the isolated LOV and HK domains nowwe intend to investigate the mechanism of light activation, that is, how thelight sensing by the LOV domain is coupled to the increase of theautophosphorylation activity of the kinase domain. Here, we will show ourrecent progress in the project, describing the crystal structure of a constructcomprising the LOV, the PAS and the connecting hinge in its dark state. Thestructure was solved at 2.74 A by molecular replacement. It is a parallel dimerwith the N-helix, both from the LOV and the PAS domain intertwined.