Study of ligand binding sites in Plasma Membrane Calcium Pump: An Azido-Ruthenium photoreactive probe as strategy for structural characterization

¨. ONTIVEROS, M 1 ; GENTILE, L 1 ; MANGIALAVORI, I, PETROSELLI G 2 , ERRA-BALSELLS R 2 , ROSSI, J P;FERREIRA-GOMES, M.

San Miguel de Tucumán

Congreso; 242. III Latin American Federation of Biophysical Societies (LAFeBS) IX IberoAmerican Congress of Biophysics XLV Reunion Anual SAB 2016; 2016

Sociedad Argentina de Biofísica

Póster, Proteins, Structure and Function Study of ligand binding sites in Plasma Membrane Calcium Pump:An Azido-Ruthenium photoreactiveprobe as strategy for structural characterization Ontiveros, M1; Gentile, L1; Mangialavori, I1,  Petroselli G2,  Erra-Balsells R2, Rossi,J P1; Ferreira-Gomes, M11Instituto de Química y FisicoquímicaBiológicas. ?Prof.Paladini?. Departamento de Química Biológica, Facultad deFarmacia y Bioquímica. Universidad de Buenos Aires.2Departamento deQuímica Orgánica, Facultad de Ciencias Exacta y Naturales. Universidad de Buenos Aires.mallkuontiveros@qb.ffyb.uba.ar The Plasma MembraneCalcium ATPase (PMCA) is a P-type ATPase that maintains the homeostasis of Ca2+in eukaryotic cells. It couples the transport of Ca2+ with thehydrolysis of ATP. The structure of PMCA is still not resolved, and only limited information is availableof ligand binding sites. The purpose of this work is to identify andcharacterize the ligand binding sites of PMCA. We synthesized azido-ruthenium(AzRu) a photoactivable reagent to obtain structural information of PMCA, whichbinds covalently and specifically to Ca2+-binding proteins afterexposure to irradiation at 290 nm1. The experiments were performedwith purified PMCA from human erythrocytes. Measurements ofphosphoenzyme in presence of AzRu showed an increase of phosphorylated intermediate in experimentalconditions that inhibit the ATPase activity. This suggests that AzRu would beaffecting the dephosphorylation of the pump.Studies of ESI-Orbitrap/MALDI-TOF-MS of PMCA with AzRu (after photolysis) showed that some peptides cannot be found,suggesting that these peptides might be related with the interaction sitesbetween AzRu and PMCA. These peptides contain histidine residues and other residues that are targets of covalent binding with the photoactivable azido group2.We analyzed the sequence homology model of PMCA on crystallographic structureof SERCA and we found a peptide related with the Mg2+site. Other peptides were analyzed because theseare in the C-terminus which is not present in SERCA. Ours results suggest that the Mg2+binding site could be involved in the interaction between PMCA and AzRu.We analyzed the structure of AzRu by MALDI/LDI-TOF and foundfragments of reagent that would be photolysis products. This result will beuseful to recognize adducts AzRu-PMCA and easily analysisof the results by mass spectrometry.[1] Israelson et al. Nature Protocols 1, 111-117, 2006[2] Bijelicet al. J of Med Chem 59, 5894-5903,2016Acknowledgementsand Support of ANPCYT, CONICET y UBACYT