GAINING INSIGHT INTO THE ACTIVATION MECHANISM OF THE RESPONSE REGULATOR NTRX FROM Brucella abortus

FERNANDEZ IGNACIO; OTERO LISANDRO; KLINKE SEBASTIAN; CARRICA MARIELA; GOLDBAUM FERNANDO

Rosario

Congreso; 50th Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2014

Brucella abortus is a pathogen that has to o vercome oxygen deficiency during the course of infection. Our group  identified the two - component system NtrY/X that is activated under reducing conditions and is important for the  induction of genes involved in the adaptation to low oxygen tension. Given t hat NtrX has not been previously  characterized, we decided to study the changes induced in its isolated receiver domain (REC) upon phosphorylation.  We solved the structure of the REC domain in the absence and presence of the phosphoryl analog beryllofluori de by  X - ray crystallography. We could determine that phosphorylation promotes dimerization of the REC domain, and  analysis of the structures showed the assembly of an interface between helix  α 4 and sheet  β 5 from neighboring  monomers. Also, major changes in the backbone of loop  β 4 α 4 were observed, allowing interactions between residues  W55 - H85, as well as repositioning of helix  α 5 . To study the relevance of H85 we obtained the REC(H85A) mutant, which is not efficiently phosphorylated and is autodephosphorylated faster than the wild - type domain. These results  present a first glimpse into the activation mechanism of NtrX, with changes in the interface between monomers and  movement of helix  α 5 as p ossible transducer events, and H85 as an important residue to determine phosphorylation  efficiency and half - life of the activated response regulator.