Nucleoside diphosphate kinase oligomerization: crystallographic and biological analyses

GOMEZ BARROSO JA; MIRANDA MR; PEREIRA CA; GARRATT RC; AGUILAR CF

Mendoza

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2012

Nucleoside diphosphate kinases (NDPK) are enzymes involved in cell nucleotide homeostasis by the interconversion of nucleoside di and tri-phosphates. TcNDPK1 is the canonical isoform of Trypanosoma cruzi, the causative agent of Chagas’Disease, and like eukaryotic NDPKs forms active hexamers. In this work we study TcNDPK1 oligomerization through molecular biology and X-ray crystallography techniques. The three dimensional structure at 3Å resolution showed that the 24 hexamers in the asymmetric unit are arranged into a helix-like oligomeric formation. The oligomerization observed in the crystalline structure was also detected in the parasite by over-expressing the NDPK gene fused to GFP, in order to reduce the intermolecular distance by the formation of weak dimers. Transgenic parasites showed a granular organization localized mainly in the anterior part of the cell that could be destabilized by changes in the intracellular salt concentration. Electron microscopy analysis indicated that these granules were filled structures without membranes. This work represents the first report of a NDPK assembled into an organized arrangement with a physiological relevance.