El rol de las regiones flanqueantes del dominio LOV del fotorreceptor LOV- histidina quinasa de Brucella

SEBASTIÁN KLINKE; FERNANDO A. GOLDBAUM; IGNACIO FERNÁNDEZ; JIMENA RINALDI

San Miguel de Tucumán

Congreso; III Reunión de Fotobiólogos Moleculares Argentinos; 2016

Grupo Argentino de Fotobiología

Light modulates the virulence of the bacterium Brucella abortus through a histidine kinase (HK) containing a light-oxygen-voltage (LOV) domain [1]. LOV domains consist of an α/β core where the cofactor FMN is bound. Light absorption causes the formation of a covalent bond between a conserved cysteine residue and the C(4a) atom from the FMN ring. While the covalent bond formation is fast (in µseconds) in all LOV proteins characterized, the recovery to the dark state varies among different LOV-containing proteins, ranging from 20-30 seconds in phototropins to hours in some prokaryotic photoreceptors. According to secondary structure predictions and based on other models, the LOV domain from Brucella LOV-HK is formed by a conserved α/β fold (core) which is flanked by an N-terminal helix (N-helix) and a C-terminal helix (J-helix). Previous work in our laboratory was performed using a construct from the LOV domain that encompasses the core region, which is an unstable dimer in solution. Its structure shows that it adopts the typical α/β PAS domain fold, consisting of a β-sheet and α-helical connector elements. NMR studies point out that the β-scaffold is a key element in the light activation [2]. Also, we reported that upon illumination the recovery to the dark state takes several hours (half-life of 5.8 hours at pH 7.0 and 35ºC) [2]. In the present work we aimed to study the role of the helices flanking the LOV core region. For that purpose, we cloned, expressed, and purified different LOV constructs containing one or both of the terminal helices. The analysis of their oligomeric state through static and dynamic light scattering indicates that the N-helix is essential for the formation of a stable dimer. We also studied dark recovery kinetics 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. Finally, we purified a construct of the LOV domain which includes both the N- and J-helices (LOV 15-155) and a mutant version insensitive to light, in which the reactive cysteine residue was replaced by serine (LOV 15-155 C69S). These proteins were submitted to a screening of crystallization solutions, obtaining different conditions in which crystals were obtained. These conditions were further optimized and the crystals will be used in diffraction experiments that are under way.