Structural characterization of the Plasma Membrane Calcium Pump by the photoreactive probe Azido-Ruthenium

ONTIVEROS, MALLKU; PETROSELLI G; ROSSI JPFC; MANGIALAVORI IRENE; ROSSI ROLANDO; GENTILE, LUCILA; ERRA-BALSELLS R; FERREIRA GOMES, MARIELA

Buenos Aires

Congreso; Protein Biophysics at the End of the World v2.0; 2017

Comité Organizador 2nd Protein Biophysics At The End of The World

The Plasma Membrane Calcium ATPase (PMCA) is a P-type ATPase that maintains the homeostasis of Ca2+ in eukaryotic cells. It couples the transport of Ca2+ with the hydrolysis of ATP. The structure of PMCA is still not resolved, and only limited information is available of ligand binding sites. The purpose of this work is to identify and characterize the ligand binding sites of PMCA. For this, we synthesized azido-ruthenium (AzRu), a photoactivable reagent designed to obtain structural information, which binds covalently and specifically to Ca2+-binding proteins after irradiation at 290 nm1. The experiments were performed with purified PMCA from human erythrocytes. Results showed that AzRu binds irreversibly to PMCA both before and after photolysis. Time-dependent assays before photolysis show that different concentrations of Ca2+ affect the rate of AzRu inhibition of Ca2+-ATPase activity, suggesting that the Ca2+ binding site could be involved in the interaction between PMCA and AzRu.Inhibition of the ATPase activity was accompanied by an increase in the phosphorylated intermediate levels, which suggests that AzRu could be blocking the dephosphorylation of the pump. Studies of ESI-Orbitrap/MALDI-TOF-MS of PMCA with AzRu (after photolysis) showed the disappearance of certain peptides containing histidine and other amino acid residues that are targets of coordination binding with the ruthenium atom2. [1] Israelson et al. Nature Protocols 1, 111-117, 2006[2] Bijelic et al. J of Med Chem 59, 5894-5903, 2016Acknowledgements and Support of ANPCYT, CONICET y UBACYT