CRYSTAL STRUCTURE OF THE NTRX RESPONSE REGULATOR AND ANALYSIS OF ITS DNA BINDING ACTIVITY

FERNÁNDEZ I; CORNACIU I; HOFFMANN G; SIEIRA R; CARRICA M; MÁRQUEZ JA; GOLDBAUM F

Mar del Plata

Congreso; SAIB - 51 Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2015

Two-componentsystems are signaling pathways, formed by a histidine kinase (HK) and aresponse regulator (RR), that allow bacteria to sense environmental cues andgenerate a response. Upon reception of a signal, the HK autophosphorylates andthen transfers the phosphate group to the RR, which is activated to perform anoutput. Our group has been studying the two-component system NtrY-NtrX fromBrucella abortus, which is involved in adaptation to low oxygen tension. The RRNtrX belongs to the NtrC family and comprises three domains: an N-terminal RECdomain, a central AAA+ domain and a C-terminal DNA binding domain (DBD). SinceNtrX is a poorly studied RR, we decided to undergo its structuralcharacterization. Using X-ray crystallography, we were able to solve thestructure of full-length NtrX. Overall, the protein crystallized as anasymmetric dimer in which the DBD adopts a conformation that is permissive forDNA binding. It is also noticeable that the presence of a long α5 helix in the REC domain sustainsan interface that is different to that reported for this truncated domain. Weconfirmed the ability of inactive NtrX to bind DNA by EMSA using the promoterof its own operon, and we also established the sequence of the DNA binding siteby footprinting. In conclusion, we provide the first crystal structure of NtrXand postulate a feedback mechanism regulating its own expression.