The ß-Scaffold of the LOV Domain of the Brucella Light-Activated Histidine Kinase is a Key Element for Signal Transduction

RINALDI J; GALLO M; KLINKE S; PARIS G; BONOMI H; BOGOMOLNI R; CICERO D; GOLDBAUM F

Cordoba

Congreso; XI Encuentro Latinoamericano de Fotoquímica y Fotobiología.; 2012

Light–oxygen–voltage (LOV) domains are blue-light-activated signalling modules present in a wide range of sensory proteins. LOV three-dimensional structures show the chromophore FMN non-covalently bound to the protein by hydrogen bonding and hydrophobic interactions, and the sulphur atom of a conserved Cys near the C4􀄮 carbon of FMN. Light absorption by the FMN cofactor results in formation of a covalent bond between the sulphur atom from the Cys residue and the C4􀄮 carbon from FMN. In most of the LOV domains this covalent bond breaks spontaneously in darkness completing the photocycle. The genomes from bacteria belonging to the Brucella genus contain a gene sequence that encodes a 463 amino acid sensory box kinase containing three domains: a LOV domain at the N-terminus (the sensory domain), a histidine kinase at the C-terminus (the output domain) and a PAS domain located between the sensory and output domains (LOV-HK). This full-length bacterial photosensory protein, expressed in E. coli and affinity purified, has a molecular weight of 55 kDa, binds FMN as a chromophore, and shows a typical LOV domain absorption spectrum with broad absorption bands in the blue (450nm) and UV-A regions (370 nm). In previous work we have shown that light modulates the virulence of the pathogenic bacterium Brucella abortus through LOV-HK. One of the striking characteristic of Brucella LOV-HK is the fact that the protein remains activated upon light sensing, without recovering the basal state in the darkness. In contrast, the light state of the isolated LOV domain slowly returns to the dark state. To gain insight into the light activation mechanism, we have characterized by X-ray crystallography and solution NMR spectroscopy the structure of the LOV domain of LOV-HK in the dark state and explored its light-induced conformational changes. The LOV domain adopts the alpha/beta PAS (PER-ARNT-SIM) domain fold and binds the FMN cofactor within a conserved pocket. The domain dimerizes through the hydrophobic beta- scaffold in an antiparallel way. Our results point to the beta-scaffold as a key element in the light activation, validating a conserved structural basis for light-to-signal propagation in LOV proteins. Finding out the interacting partner surface of the LOV domain beta-scaffold in Brucella LOV-HK will reveal further details of the molecular mechanisms of light activation. To answer this question, we are trying to solve the crystallographic structure of the different domains that build up the whole LOV-HK protein. Several constructs are being studied at the moment (HK,LOV-Jalpha-PAS, LOV-Jalpha, point mutants, etc), including complex structures with the two putative response regulators of this light activated two-component system.