Structural studies on the C-terminal domain of human PMCA1b

Plasma Membrane Calcium ATPases (PMCAs) are P-type pumps involved in calcium homeostasis. Their 3D structures are still unknown but a possible topology has been predicted. PMCAs are predicted to have a cytosolic N-terminal domain, a cytosolic C-terminal domain (regulatory domain), ten transmembrane segments and two cytosolic loops called transduction domain and catalytic domain that connect the 2nd and the 3rd, the 4th and the 5th transmembrane segments respectively. Several mechanisms are responsible of their activation: interaction with Ca2+-calmodulin, interaction with acidic phospholipids and fatty acids, phosphorylation with kinases A and C, proteolysis by calpain and oligomerization. All activation mechanisms decrease the Km values, in particular the oligomerization brings this value around at the value of cytosolic calcium concentration present in the resting cells (50-100 nM). The C-terminal domain is a structural motif that distinguishes PMCAs from all other P-type pumps. It is also the target of all activators and activation mechanisms. In this study we have described the secondary structure and tertiary structure at low resolution of the C-terminal regulatory domain of the human PMCA isoform 1b. We have found that the domain forms aggregates by intermolecular interactions. Moreover, we have studied the reversibility of the oligomerization process and found the best conditions to stabilize the C-terminal domain in the monomeric form. These conditions imply the presence of the lipid mimetic SDS at critical micellar concentration. A structural reconstruction based on Small Angle Neutron Scattering experiments provides a low resolution structure where the C-terminal domain has an hourglass shape. The central cross section compatible with that of an ?-helix. This part could correspond at the ?-helix of the C28W calmodulin binding region while the downstream and upstream regions could be random coil as also predicted by PSIpred. Binding experiments between the C-terminal domain and the Ca2+- calmodulin have been carried out. The aim was to study whether in a phospholipid mimetic system necessary to stabilize the monomeric form, such as sodium dodecyl sulphate, this domain can still interact with calmodulin. The phospholipid mimetic system that stabilize the domain in the monomeric form prevent its binding with Ca2+-calmodulin. The results suggest that a different aggregation state of the PMCAs exist in diverse membrane rafts: membrane rafts rich in uncharged or zwitterionic phospholipids could contain PMCAs in oligomeric form while membrane rafts rich in acidic phospholipids could contain PMCAs in monomeric form.