A New Conformation in Sarcoplasmic Reticulum Calcium Pump and Plasma
Membrane Ca2+ Pumps Revealed by a Photoactivatable Phospholipidic
Probe |
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Authors: | Irene Mangialavori Ana Mar??a Villamil Giraldo Cristina Marino Buslje Mariela Ferreira Gomes Ariel J Caride and Juan Pablo F C Rossi |
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Institution: | ‡Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, CONICET, Junín 956 (1113) Buenos Aires, Argentina and the §Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905 |
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Abstract: | The purpose of this work was to obtain structural information about
conformational changes in the membrane region of the sarcoplasmic reticulum
(SERCA) and plasma membrane (PMCA) Ca2+ pumps. We have assessed
changes in the overall exposure of these proteins to surrounding lipids by
quantifying the extent of protein labeling by a photoactivatable
phosphatidylcholine analog
1-palmitoyl-2-9-2′-125I]iodo-4′-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine
(125I]TID-PC/16) under different conditions. We determined the
following. 1) Incorporation of 125I]TID-PC/16 to SERCA decreases
25% when labeling is performed in the presence of Ca2+. This
decrease in labeling matches qualitatively the decrease in transmembrane
surface exposed to the solvent calculated from crystallographic data for SERCA
structures. 2) Labeling of PMCA incubated with Ca2+ and calmodulin
decreases by approximately the same amount. However, incubation with
Ca2+ alone increases labeling by more than 50%. Addition of C28, a
peptide that prevents activation of PMCA by calmodulin, yields similar
results. C28 has also been shown to inhibit ATPase SERCA activity.
Interestingly, incubation of SERCA with C28 also increases
125I]TID-PC/16 incorporation to the protein. These results suggest
that in both proteins there are two different E1
conformations as follows: one that is auto-inhibited and is in contact with a
higher amount of lipids (Ca2+ + C28 for SERCA and Ca2+
alone for PMCA), and one in which the enzyme is fully active (Ca2+
for SERCA and Ca2+-calmodulin for PMCA) and that exhibits a more
compact transmembrane arrangement. These results are the first evidence that
there is an autoinhibited conformation in these P-type ATPases, which involves
both the cytoplasmic regions and the transmembrane segments.Although membrane proteins constitute more than 20% of the total proteins,
the structure of only few of them is known in detail. An important group of
integral membrane proteins are ion-motive ATPases. These proteins belong to
the family of P-type ATPases, which share in common the formation of an
acid-stable phosphorylated intermediate as part of its reaction cycle.
Crystallographic information is available for a few members of this family.
There are several crystal structures of the Ca2+ pump of
sarcoplasmic reticulum
(SERCA)2 revealing
different conformations
(1–5),
and recently, crystal structures of the H+-ATPase
(6) and of the Na,K-ATPase were
reported as well (7).We are interested in obtaining structural information about the plasma
membrane calcium pump (PMCA). This pump is an integral part of the
Ca2+ signaling mechanism
(8). It is highly regulated by
calmodulin, which activates this protein by binding to an auto-inhibitory
region and changing the conformation of the pump from an inhibited state to an
activated one (8,
9). Crystallization of PMCA is
particularly challenging because there is no natural source from which this
protein can be obtained in large quantities. Moreover, the presence of several
isoforms in the same tissue further complicates efforts to obtain a
homogeneous sample suitable for crystallization.Information about the structure and assembly of the transmembrane domain of
an integral membrane protein can also be obtained from the analysis of the
lipid-protein interactions. In this work, we have used a hydrophobic
photolabeling method to study the noncovalent interactions between PMCA and
the surrounding phospholipids under different experimental conditions that
lead to known conformations. We employed the photoactivatable
phosphatidylcholine analog
1-palmitoyl-2-9-2′-125I]iodo-4′-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine
(125I]TID-PC/16) that has been previously used to analyze
lipid-protein interfaces
(10–12).
This reagent is located in the phospholipidic milieu, and upon photolysis it
reacts indiscriminately with its molecular neighbors. It is thus possible to
directly analyze the interaction between a membrane protein and lipids
belonging to its immediate environment
(13–15).
By measuring the amount of labeling of SERCA in conditions that promote
conformations for which there are well resolved crystal structures, we were
able to validate this photolabeling approach as a convenient tool for
analyzing conformational changes within transmembrane regions. Furthermore,
using this technique on PMCA and comparing the results obtained for SERCA, we
were able to draw structural conclusions about these proteins under activated
and inhibited states. |
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Keywords: | |
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