STRUCTURAL AND FUNCTIONAL STUDIES OF THE NTRX RESPONSE REGULATOR, A DIMERIC ATP BINDING PROTEIN

CARRICA MC; GOLDBAUM FA; CORNACIU I; MÁRQUEZ JA; FERNÁNDEZ I; UCHIKAWA E

Cordoba

Congreso; 52th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2016

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 uponperception of a stimulus and transfers the phosphoryl group to the secondcomponent, the response regulator (RR), which modulates gene transcription. Ourgroup has been interested in a TCS formed by the HK NtrY and the RR NtrX, whichhas 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 studiesallowed us to obtain the crystal structure of the full-length protein and toanalyze the DNA binding. In this opportunity, we have examined some characteristicsrelated to the AAA+ domain in order to gain insights into how NtrX works. Wefound that this RR is able to bind ATP but it cannot hydrolyze the nucleotide.Furthermore, we solved the structures obtained by soaking NtrX crystals withATP and ADP, and describe the binding pocket. Also, we found that NtrX is adimer in solution and that it does not undergo further oligomerization as aconsequenceof phosphorylation or nucleotide binding. Finally, we have developed apreliminary design for an in-vivo assay with Caulobacter crescentusto performstructure-function studies to identify important residues in NtrX mechanism ofaction.