Structural and functional studies of the NtrX response regulator, a dimer ATP binding protein

IRINA CORNACIU; JOSÉ ANTONIO MÁRQUEZ; MARIELA CARRICA; FERNANDO GOLDBAUM; IGNACIO FERNANDEZ; EMIKO UCHIKAWA

Cordoba

Congreso; LII Reunión de la Sociedad Argentina de Bioquímica y Biología Molecular (SAIB); 2016

SAIB

Bacteria need to adapt to environmental changes in order to survive. Among the mechanisms employed for this task are the two-component systems (TCS). They are formed by a histidine kinase (HK) that autophosphorylates upon perception of a stimulus and transfers the phosphoryl group to the second component, the response regulator (RR), which modulates gene transcription.Our group has been interested in a TCS formed by the HK NtrY and the RR NtrX, which has been implicated in the detection of low oxygen tension in Brucella abortus. NtrX has a REC, a central AAA+, and a DNA binding domain. Previous studies allowed us to obtain the crystal structure of the full-length protein and to analyze the DNA binding. In this opportunity, we have examined some characteristics related to the AAA+ domain in order to gain insights into how NtrX works. We found that this RR is able to bind ATP but it cannot hydrolyze the nucleotide. Furthermore, we solved the structures obtained by soaking NtrX crystals with ATP and ADP, and describe the binding pocket. Also, we found that NtrX is a dimer in solution and that it does not undergo further oligomerization as a consequence of phosphorylation or nucleotide binding.Finally, we have developed a preliminary design for an in-vivo assay with Caulobacter crescentus to perform structure-function studies to identify important residues in NtrX mechanism of action.