STRUCTURAL AND FUNCTIONAL DIFFERENCES BETWEEN SARCOPLASMIC RETICULUM CALCIUM PUMP (SERCA) AND PLASMA MEMBRANE CALCIUM PUMP (PMCA) REACTION CYCLE INTERMEDIATES.

SAFFIOTI1, NICOLÁS ANDRÉS; DE SAUTU, MARILINA1; MANGIALAVORI, IRENE CECILIA1; FERREIRA-GOMES, MARIELA1; ROSSI ,ROLANDO1; BERLIN, JOSHUA2 AND ROSSI; JUAN PABLO1

New Orleans

Congreso; 61 st Biophysical Society Meeting; 2017

Biophysical Society

Summary: Control/Tracking Number: 17-A-557-BPS Activity: Abstract Current Date/Time: 10/7/2016 2:20:19 PM  STRUCTURAL AND FUNCTIONAL DIFFERENCES BETWEEN SARCOPLASMIC RETICULUM CALCIUM PUMP (SERCA) AND PLASMA MEMBRANE CALCIUM PUMP (PMCA) REACTION CYCLE INTERMEDIATES. Author Block Nicolás Andres Saffioti1, Marilina de Sautu1, Irene C. Mangialavori1, Mariela Ferreira Gomes1, Rolando C. Rossi1, Joshua R. Berlin2, JUAN PABLO F. ROSSI1. 1DEPT. QUÍMICA BIOLOGICA, IQUIFIB, CAPITAL FEDERAL, BUENOS AIRES, Argentina, 2Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, CAPITAL FEDERAL, BUENOS AIRES, NJ, USA. Abstract: SERCA and PMCA belong to the P-ATPases family. SERCA?s structure and function have been widely characterized whereas PMCA has a relatively high sequence identity with SERCA but its structure is less known. Although both proteins pump Ca2+ out of the cytoplasm of cells, their kinetic properties are very different. To understand this on the basis of differences in their structure, we employed fluoride complexes with beryllium, aluminium or magnesium, which stabilize different analogues of the phosphorylated intermediates in P-ATPases, although they have never been tested on PMCA. To study the protein structure we employed the hydrophobic photoactivatable probe 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) which labels the transmembrane domains of these proteins and the fluorescent probe 2´,3´-O-(2,4,6-Trinitrophenyl)adenosine-5´-triphosphate (TNP-ATP) which binds to the nucleotide binding-domain of both pumps. Results show that BeF3- and AlF4- inhibit PMCA activity at micromolar concentrations, whereas millimolar concentrations of Mg2+ and F- are required to achieve that effect. TID labeling in different conformations of SERCA correlates well with the protein surface exposed to the bilayer calculated from crystallographic models, with less labeling in the presence of calcium. Irrespectively of the presence of metal-fluoride complexes, TID labeling of PMCA only decreases when the pump is incubated with calcium and calmodulin, indicating a lower exposure of the transmembrane region. When incubated with metal-fluoride complexes, TNP-ATP increases its quantum yield when it is bound to SERCA but decreases by a half when it is bound to PMCA. Our results indicate that calmodulin binding to PMCA allows conformational changes in the transmembrane region similar to those observed in SERCA in presence of calcium, but the nucleotide binding domain behaves very differently when these proteins are in a phosphorylated-like state. With grants from ANPCYT, CONICET and UBACYT