INVESTIGADORES
ROSSI Juan Pablo Francisco
congresos y reuniones científicas
Título:
Conformational changes of the transmembrane domain of the Plasma Membrane Calcium Pump by calmodulin and acidic phospholipids
Autor/es:
M. FLORENCIA PIGNATARO, ANA VILLAMIL GIRALDO, MARIELA FERREIRA-GOMES, JUAN PABLO ROSSI AND IRENE MANGIALAVORI
Lugar:
Salta
Reunión:
Congreso; SAB 2010 Workshop CeBEM-Protein Society Meeting; 2010
Institución organizadora:
SAB-Protein Society
Resumen:
Conformational changes of the transmembrane domain of the Plasma
Membrane Calcium Pump by calmodulin and acidic phospholipids. M.
Florencia Pignataro, Ana Villamil Giraldo, Mariela Ferreira-Gomes, Juan Pablo Rossi and Irene Mangialavori.
The
Plasma Membrane Calcium Pump (PMCA) is an integral membrane protein, from the
family of the P-ATPases. The reaction cycle of these proteins is characterized
for a phosphorylated intermediary. Its ATPase activity is enhanced by several
modulators among which calmodulin (CaM) and acidic phospholipids appear to be
the most effective ones. Whenever Ca2+ intracelular concentration rises,
calmodulin binds to the C-terminal cytoplasmatic domain, generating a
conformational change that releases the protein from its previous state of
auto-inhibition. The acidic phospholipids binding site could be located in the
cytoplasmatic loop that connects the transmembrane segments 2 and 3 (TM2 y
TM3). Some author, suggest the existence of a second site in the C-terminal
domain near to the CaM binding site, that would prevent CaM mediated activation
when acidic phospholipids are present. The purpose of this work was to obtain
structural information about conformational changes of PMCA related to the
activation by calmodulin (CaM) and phosphatidic acid (PA). The transmembrane
region was studied by measuring the specific incorporation of the
photoactivatable phosphatidylcholine analog, [125I]TID-PC/16. Previously, this
strategy allowed us to demonstrate the existence of a transmembrane inhibited
conformation in the presence of Ca2+ (E1I) and an active conformation (E1As) in
the presence of activators that expose a different surface to surrounding
phospholipids [1, 2]. The cytoplasmatic region was studied by the access of two
proteases to their sites of cleavage and measuring FRET between the labeled
pump with eosin isothiocyanate (EITC-PMCA) and fluorescent
phosphatidylethanolamine analog (RhoPE). 1) FRET experiments would be
suggesting that in the conformation E1I (obtained with Ca2+ alone) the
cytoplasmatic domain containing the ATP binding site (where PMCA is labeled
with EITC) is closer to the membrane. Addition of CaM, to form E1
activated (E1A), results in this domain located in a position
further away from the membrane. Regarding the experiment in which we measured
FRET efficiency between EITC-PMCA and RhoPE with increasing amounts of PA, it
will be said that, as expected, increasing the amount of lipids decreased the
fluorescence because of RhoPE dilution. However, the decrease in FRET was the
same regardless of the nature of the phospholipid added (phosphatidylcholine,
phosphatidylethanolamine, or phosphatidic acid). This indicates that during the
activation by acidic lipids, the cytoplasmic domain which contains the ATP
binding site does not move significantly farther away from the membrane. This
strongly differs from the results obtained with the enzyme activated by CaM. With grants
of ANPCYT, CONICET and UBA.