Characterization of a PIB-ATPase from the psychrophilic bacteria Bizionia argentinensis

ALVARO RECOULAT; F LUIS GONZÁLEZ FLECHA

Edinburgo

Congreso; 19th International Union of Pure and Applied Biophysics (IUPAB) and 11th European Biophysical Societies' Association (EBSA) Congress; 2017

IUPAB

P IB -ATPases are integral membrane proteins which arewidespread distributed in nature, and actively transportheavy metal ions through cell membranes coupled to thehydrolysis of ATP. In this work we isolate and character-ize a putative P1B-type ATPase from the Antarctic bac-terium Bizionia argentinensis (BaCopA). This protein wascloned and expressed in S. cerevisiae as a GFP-fusion pro-tein. Membrane proteins were solubilized with sodium de-oxycholate and BaCopA purified by IMAC chromatography.The obtained protein run as a single band on SDS PAGE,and display ATPase and phosphatase activity with a max-imum around 20°C and pH 8.0. The ATPase activity isenhanced by Mg 2+ and phospholipids, and inhibited by theCu(I) chelator BCS. An homology structural model was ob-tained using the resolved structure of L. pneumophila CopAas template (PDB: RFU3). The structural alignment showa high degree of similarity, with the typical topological pat-tern of P 1B-1 ATPases: an N-terminal Heavy Metal Asoci-ated domain (residues 4-62), an actuator domain (residues296-509), an ATP binding domain (residues 522-741), and atransmembrane domain composed of 8 transmembrane he-lices including the characteristic residues involved in Cu(I)binding and translocation.With grants from UBA, CONICET and ANPCyT.