PMCA conformational changes in phosphorylated intermediaries

ROSSI JUAN PABLO; BERLIN, JOSHUA; SAFFIOTI, NICOLÁS ANDRÉS; MANGIALAVORI, IRENE CECILIA

San Miguel de Tucumán

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

Latin American Federation of Biophysical Societies (LAFeBS)

Plasma membrane calcium pump (PMCA) plays a key role in cellularcalcium homeostasis by pumping calcium ions outside the cytoplasm.PMCA has a relatively high sequence identity with other P-ATPases likethe sarcoplasmic reticulum calcium pump (SERCA). However PMCAstructure is less known and has particular properties as presence of anautoinhibitory calmodulin binding site in its C-terminal segment.In order to understand how calmodulin binding alters PMCA structure,we employed fluoride complexes with beryllium, aluminium ormagnesium, which are proposed to stabilize different analogues of thephosphorylated intermediates in P-ATPases. To study the PMCA structurewe employed the photoactivable probe 3-(trifluoromethyl)-3-(miodophenyl)diazirine(TID) which labels the transmembrane domainsof 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 labeling show that PMCA surfaceexposed to the bilayer only decreases in presence of both calcium andcalmodulin. However this change also occurs upon fluoride complexesbinding. On the other hand upon addition of metal-fluoride complexes,TNP-ATP bound to PMCA decreases its quantum yield by a half withoutchanging the affinity of the probe for the protein. This is consistentwith a more exposure to solvent of ATP binding site. The conformationalchange occurs even when the pump is in presence of calcium andcalmodulin.These results indicate that conformational changes in the transmembraneregion of PMCA are restricted in absence of calcium and calmodulin,but even so the nucleotide binding domain is able to hingeupon phosphorylation. By homology modeling of PMCA sequenceon SERCA crystallographic structures, we evaluate the implications ofthese results on PMCA structure model and how the C-terminal domainrestricts conformational changes in this protein.With grants of CONICET, ANPCYT and UBA.