PMCA conformational changes in phosphorylated intermediates

NICOLÁS SAFFIOTI; JOSUA BERLIN; JUAN PABLO ROSSI; IRENE MANGIALAVORI

Congreso; Reunión conjunta XLV Reunión Anual de la Sociedad Argentina de Biofísica (SAB) / III LAFeBS / X IberoAmerican Congress of Biophysics.; 2016

Plasma membrane calcium pump (PMCA) plays a key role in cellular calcium homeostasis by pumping calcium ions outside the cytoplasm. PMCA has a relatively high sequence identity with other P-ATPases like the sarcoplasmic reticulum calcium pump (SERCA). However PMCA structure is less known and has particular properties as presence of an autoinhibitory 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 or magnesium, which are proposed to stabilize different analogues of the phosphorylated intermediates in P-ATPases. To study the PMCA structure we employed the photoactivable probe 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) which labels 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 labeling show that PMCA surface exposed to the bilayer only decreases in presence of both calcium and calmodulin. However this change also occurs upon fluoride complexes binding. On the other hand upon addition of metal-fluoride complexes, TNP-ATP bound to PMCA decreases its quantum yield by a half without changing the affinity of the probe for the protein. This is consistent with a more exposure to solvent of ATP binding site. The conformational change occurs even when the pump is in presence of calcium and calmodulin.These results indicate that conformational changes in the transmembrane region of PMCA are restricted in absence of calcium and calmodulin, but even so the nucleotide binding domain is able to hinge upon phosphorylation. By homology modeling of PMCA sequence on SERCA crystallographic structures, we evaluate the implications of these results on PMCA structure model and how the C-terminal domain restricts conformational changes in this protein. With grants of CONICET, ANPCYT and UBA.