Study of a two-component system activated by light in Brucella abortus (Póster)

GABRIELA SYCZ; FERNANDO A. GOLDBAUM; SEBASTIÁN KLINKE; LISANDRO OTERO; IGNACIO FERNÁNDEZ; JIMENA RINALDI

Hyderabad

Congreso; 24th Congress and General Assembly of the International Union of Crystallography; 2017

International Union of Crystallography (IUCr)

Two-component signal transduction systems (TCSs)are modules that allow bacteria to rapidly adapt to changing environmentalconditions. In the most common case, they are formed by a sensor histidinekinase (HK) which, upon sensing of an external signal, autophosphorylates at a conserved histidineresidue and then transfers the phosphoryl group to a conserved aspartateresidue in a cognate response regulator (RR). The latter protein undergoesstructural changes that are able to modify gene expression by directly bindingto DNA, catalyze metabolic reactions or alter protein-protein interactions. Thepathogenic bacterium Brucella abortus, the causative agent of the diseasebrucellosis, bears a particular two-component system formed by a dimeric cytoplasmicthree-domain blue-light sensor HK (LOV-PAS-HK) and two monomeric RRs calledPhyR and LovR. The activation of this HK has been shown to increase thevirulence of this pathogen. With the goal of understanding at the atomic levelthe activation and signal transduction events of this system, we aimed to solvethe three-dimensional structures of these proteins by means of X-raydiffraction. The core of the blue-light sensor FMN-binding LOV domain wascrystallized and its structure solved at 1.64 Å resolution in the dark. It adoptsthe alpha/beta PAS domain fold and presents a hydrophobic central beta-scaffoldthat interacts in one face with an FMN molecule and in the other with a neighboringmonomer forming an unexpected antiparallel homodimer. This beta-scaffolddestabilizes upon light exposure and therefore was proposed as a key element inthe signal transduction mechanism [1]. Interestingly, we were also able todetermine the structure of a construct comprising the LOV core domain plus anN-terminal capping helix at 2.34 Å resolution (N-LOV), observing now theexpected parallel dimerization nature of the protein. This structure let usunderstand at the atomic level the important contribution of this N-terminalelement in the stabilization of the quaternary structure and its photochemicalbehavior. This parallel arrangement has been recently confirmed with thedetermination of the N-LOV-PAS structure at 2.74 Å resolution in the lab, whichholds a long connecting alpha-helical element between both globular domains. Additionally,we were able to solve the crystal structure of the isolated HK domain at 2.51 Åresolution by sulfur SAD in a challenging procedure, due to low sequenceidentity available models for MR, the low symmetry P2(1) space group present andthe existence of four copies of the molecule in the 108-kDa asymmetric unit(AU) [2]. Interestingly, the HK structure presents two different dimeric assembliesin the AU, which allowed us to propose a mechanism of activation [3]. Tofinish, we were also able to determine the structure of the PhyR RR at 2.05 Åresolution. Efforts are underway to obtain the structure of the full LOV-PAS-HKprotein as well as HK-RR complexes. All these protein structures, together withspectroscopic, activity and biophysical assays, allowed us a betterunderstanding of this crucial system for the pathogenicity of Brucella.