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From Proteopedia

Revision as of 12:04, 2 January 2015

Introduction

All living organisms depend on P-type ATPase to pump cation across the membrane. They play a fundamental role in their metabolism and physiology. Ca2+ ATPase, a P-type ATPase, transports calcium ions across the membrane against a concentration gradient. These pumps clear cytoplasm of the second messenger, the calcium. It's very important to keep a low concentration of calcium in the cell for a good cell signaling. We can found the PMCA (Plasma Membrane Ca2+ ATPase) which remove the calcium from the cell and the SERCA (Sarcoplasmic Endoplasmic Reticulum Ca2+ ATPase) which pumps calcium into the endoplasmic reticulum. The hydrolysis of one adenosine triphosphate (ATP) is essential for the functioning of the pump and the transport of one calcium ion.

Structural Highlights

The calcium ATPase is a protein composed of 1001 aminoacids. The protein is very rich in . It contains 10 transmembrane alpha helices, and three of them line a channel that spans the lipid bilayer and that allows calcium to pass through membranes. It also contains too cytoplasmic loops between the thransmembrane helices. When the protein isn’t phosphorylated, two of the transmembrane helices are disrupted and form a cavity that can bind two molecules of calcium.

The transmembrane portion of the protein contains the channel that span the lipid bilayer, and the calcium binding cavity.

The two cytoplasmic loops form three separate domains. The nucleotide binding domain (N) contains the site where ATP binds to the protein. The phosphorylation domain (P) contains an Aspartate residue (Asp 351) that can be phosphorylated. Finally, the actuator domain (A) is involved in the transmission of major conformational changes. The phosphorylation and the nucleotide binding domains form the catalytic site of the protein.

Ligand and Interaction

The architecture of calcium ATPase (determined by X-Ray crystallography) allow to understand mechanisms by which the energy of ATP is coupled to the calcium transport across a membrane.

The first step of the calcium pump catalytic cycle is the cooperative binding of two calcium ions in the calcium binding cavity. Then, ATP binds to the ATP binding site (nucleotide binding domain) and transfers its γ-phosphate to the aspartic acide 351 (phosphorylation domain). That creates a acid-stable aspartyl phosphate intermediate. The phosphorylation of Asp351 allows a large conformational changes in cytoplasmic domains: the nucleotide binding domain and the phosphorylation domain are brought into close proximity. This rearrangement causes a 90° rotation of the activator domain, which leads to a rearrangement of the transmembrane helices. This rearrangement alters the affinity of the protein for the calcium and disrupts the calcium binding cavity. Calcium is released in the lumen of the endoplasmic reticulum/Golgi Apparatus or outside the cell. After releasing calcium, two protons are bound to the transport sites (charges compensation) and the aspartyl phosphate is hydrolyzed to complete the cycle.

In the cytoplasm, the ATP binds to the cytosolic loop that connects transmembrane domains four and five (ATP binding site). It transfers its γ-phosphate to the aspartic acide 351 (phosphorylation site) and creates a acid-stable aspartyl phosphate intermediate. The binding of ATP is initiated by the cooperative fixing of two calcium ions to the transport site. The phosphorylation of Asp351 allows a large conformational changes in cytoplasmic domains which closes the ion gates from the cytoplasm and alters the affinity of the protein for the calcium. After releasing calcium (in the lumen of cytoplasm or out side the cell), two protons are bound to the transport sites (charges compensation) and the aspartyl phosphate is hydrolyzed to complete the cycle.

To sum up, calcium pumps have two conformations, E1 and E2. These two conformations are characterized by different specificity for ion binding. When the pump is in the E1 state, it has high calcium affinity and interacts with calcium at one side of the membrane. In the E2 state, the enzyme has a lower calcium affinity and that leads to the release of the ion at the opposite side. E1 has the calcium binding site oriented toward the cytoplasm. E2 has the calcium binding site oriented toward the lumen of the endoplasmic reticulum or toward the extracellular background.

Biological Function and Localisation

Regulations

There are different kind of calcium ATPase regulations. For example, the phospholamban (PLN or PLB) is a membrane protein that regulates the calcium pump in cardiac muscle and skeletal muscle cells. This small phosphoprotein is a pentamer. The phospholamban can be phosphorylated at three distinct sites by various protein kinases (PKA, PKC, CamK...). The phosphorylation state is mediatedthrough beta-adrenergic stimulation. In unphosphorylate state, the phospholamban inhibits the activity of calcium pump in cardiac and skeletal muscle cells by decreasing the apparent affinity of the ATPase for calcium. The phosphorylation of the protein results in the dissociation of the protein from the ATPase. The phosphoprotein binds just downstream of the active ATPase site (asp351). The activity of the calcium pump is also regulated by by calmodulin, acidic phospholipids and phosphorylation by kinases A and C. Most of the activation mechanisms implicate the C-terminal region of the pump containing the high affinity calmodulin binding domain, which is involved in the autoinhibition of the pump.

Dysfunctions and Diseases

References

David H.MacLennan, William J.Rice and N. Michael Green, 1997 - The Mechanism of Ca2+ Transport by Sarco(Endo)plasmic Reticulum Ca2+-ATPases - The Journal of Biological Chemistry, p.272, 28815-28818

Marianela G.Dalghi, Marisa M.Fernández, Mariela Ferreira-Gomes, Irene C.Mangialavori, Emilio L.Malchiodi, Emanuel E.Strehler and Juan Pablo F.C.Rossi, 2013 - Plasma Membrane Calcium ATPase Activity Is Regulated by Actin Oligomers through Direct Interaction - The Journal of Biological Chemistry, p.288, 23380-23393

Marisa Brini and Ernesto Carafoli, 2010 - The Plasma Membrane Ca2+ ATPase and the Plasma Membrane Sodium Calcium Exchanger Cooperate in the Regulation of Cell Calcium - Cold Spring Harbor Perspectives in Biology

Marisa Brini and Ernesto Carafoli, 2009 - Calcium Pumps in Health and Disease- Physiological Reviews

Thomas D.Pollard and William C. Earnshaw, - Membrane, structure and function - Cell Biology (second edition), p.133-136

Benjamin Lewin, 2007 - Cells - Jones & Bartlett Learning.