Conformational changes produced by ATP binding to the plasma membrane calcium pump

MANGIALAVORI IC, FERREIRA-GOMES MS, SAFFIOTI NA, GONZALEZ-LEBRERO RM, ROSSI RC, ROSSI JP.

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Lugar: Bethesda, Maryland; Año: 2013

0021-9258

Background : Plasma membrane calcium ATPase (PMCA) reaction cycle is associated to conformational changes. Results : We identified different conformations after the association of Ca 2+ , ATP and vanadate to PMCA. Conclusion : PMCA forms a stable complex with Ca 2+ and vanadate ; ATP can bind to all pump conformations. Significance : This study found a new intermediate in the PMCA reaction cycle; al l the intermediates interact with ATP. The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterising conformational changes associated with calcium, ATP and vanadate binding to purified PMCA . This was accomplis hed by study ing t he exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog [ 125 I]TID - PC/16 to the protein. ATP could bind to the different vanadate - bound states of the enzyme either i n the presence or in the absence of Ca 2+ with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog TNP - ATP . To assess the conformational behaviour of the Ca 2+ binding domain we also studied the occlusion of Ca 2+ , both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and /or ATP . This allowed the development of a model that describes the transport of Ca 2+ and its relation with ATP hydrolysis. This is the first approach which uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E 1 -E 2 model devised using kinetics