Conformational changes by ATP binding in the plasma membrane calcium pump

SAFFIOTI NICOLÁS; FERREIRA GOMES MARIELA; ROSSI JUAN PABLO F. C. ; MANGIALAVORI IRENE C.

Tucuman

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofísica

Plasma membrane calcium pump (PMCA) is a P-ATPase that transports Ca2+ against the electrochemical gradient from the cytoplasm to the outer cellular medium. The principal modulator of PMCA is calmodulin which increase its Ca2+ affinity and the maximum transport rate. The reaction cycle of the pump can be described by the E1-E2 model. The first step is the Ca2+ binding to the protein, drawing it to the E1 conformation. The ATP binding and the later autophosphorylation elicit PMCA to E1P. In the next step the pump reaches the E2P conformation, which allows the liberation of Ca2+ to the cytoplasm. However it has been described that besides the catalytic role of ATP; there is another binding site for the nucleotide with a regulatory role associated with the E2 conformation. The aim of this work is to study the ATP binding to purified PMCA and the associated conformational changes, using the fluorescent analogue TNP-ATP. We also tested the conformational changes associated with the presence of calmodulin. Our results show that: (1) the TNP-ATP binds to the PMCA (2) The environment of the ATP binding domain changes in the different conformations of the PMCA. (3) The PMCA affinity for the TNP-ATP depends on the PMCA conformation (4) The PMCA affinity for TNP-ATP is much lower in E2 conformation than in E1, suggesting the existence of an ATP low-affinity binding site(s). [1] Shmuel Muallem and Steven J. D. Karlish.(1983) J.Biol.Chem. 258 159-175 [2] Suzuki, H., Kubota, T. ,Kubo ,K. ,Kanazawa, T. (1990) Biochemistry. 29, 7040-7045 With grants of ANPCYT, CONICET, UBACYT y NIH.