PMCA conformational changes in phosphorylated intermediates.

SAFFIOTI, NICOLÁS1, BERLIN JOSHUA2, ROSSI JUAN PABLO1 AND MANGIALAVORI IRENE CECILIA1

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

PMCA conformational changes in phosphorylatedintermediariesSaffioti,Nicolás Andrés; Ferreira-Gomes, Mariela; Berlin,Joshua; Rossi, Juan Pablo; Mangialavori, Irene CeciliaPlasma membrane calcium pump (PMCA) plays a key rolein cellular calcium homeostasis by pumping calcium ions outside the cytoplasm. PMCAhas a relatively high sequence identity with other P-ATPases like thesarcoplasmic reticulum calcium pump (SERCA). However PMCA structure is lessknown and has particular properties as presence of an autoinhibitory calmodulinbinding site in its C-terminal segment.In order to understand how calmodulin binding altersPMCA structure, we employed fluoride complexes with beryllium, aluminium or magnesium,which are proposed to stabilize different analogues of the phosphorylatedintermediates in P-ATPases. To study the PMCA structure we employed thephotoactivable probe 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) whichlabels the transmembrane domains of 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 ATPases. TID labelingshow that PMCA surface exposed to the bilayer only decreases in presence ofboth calcium and calmodulin. However this change also occurs upon fluoridecomplexes binding. On the other hand upon addition of metal-fluoride complexes,TNP-ATP bound to PMCA decreases its quantum yield by a half without changingthe affinity of the probe for the protein. This is consistent with a moreexposure to solvent of ATP binding site. The conformational change occurs evenwhen the pump is in presence of calcium and calmodulin.These results indicate that conformational changes inthe transmembrane region of PMCA are restricted in absence of calcium andcalmodulin, but even so the nucleotide binding domain is able to hinge uponphosphorylation. By homology modeling of PMCA sequence on SERCAcrystallographic structures, we evaluate the implications of these results onPMCA structure model and how the C-terminal domain restricts conformationalchanges in this protein. With grants of CONICET, ANPCYT and UBA.