Sandbox Reserved 820

This Sandbox is Reserved from 06/12/2018, through 30/06/2019 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1480 through Sandbox Reserved 1543.

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<StructureSection load='2vaf' size='450' side='right'caption='Crystal Structure of Human Cardiac Calsequestrin, (PDB code 2vaf) ' scene='56/568018/General_structure/4' > Calsequestrin-2 (or CASQ2) is the soluble Ca²⁺ binding protein in the sarcoplasmic reticulum lumen of the cardiac muscle cells. CASQ2 could be either in a monomeric, homodimeric, or homooligomeric chain form depending on its bounds with Ca²⁺. Mutations of CASQ2 are involved in cardiac diseases such as Catecholaminergic Polymorphic Ventricular Tachycardia.

Calsequestrin in the calcium cycle of myocyte contraction

Biological role

The contractions of cardiac myocytes are triggered by the increase of calcium concentration in the cytosol. This phenomenon is highly controlled at several levels. First the calcium is stocked in a cell compartment called the sarcoplasmic reticulum. Then the release of calcium in the cytosol is dependent of the myocytes membrane depolarization. Finally the release of calcium is extremely brief, as soon as the depolarization is over, the calcium is actively pumped in the sarcoplasmic reticulum.

The calsequestrin 2 plays a major role here, because it helps the release of the calcium in the cytosol while the membrane depolarization occurs and traps the calcium inside the lumen of the sarcoplasmic reticulum. It is also good to notice that a huge release of calcium in the cytosol would be lethal to the cell, since the calcium would precipitate with the free phosphate groups.

Structure

Monomere Structure

Each monomer is divided in 3 thioredoxin domains (TRX): the N-term, the middle and the C-term domains. Each of these has a regular structure: a 5 strands beta sheet core surrounded by 4 alpha helices. Usually these domains are involved in redox phenomena, which lead to disulfide bounds creation. Here these domains are inactive but play an important role in the polymerization of CASQ2.

Polymer Structure

Inside the sarcoplasmic reticulum lumen, CASQ2 polymerizes to form homodimers, homotetramers and homooligomers. There are two types of dimerisation: the front-to-front form and the back-to-back form. The front-to-front form is stabilized by intermolecular interactions between the α2 helix of the domain I of each calsequestrin. The intermolecular salt bridges are between Glu 55 and Lys 49. This dimerisation induces the formation of an electronegative pocket which involves these amino acids: for the first calsequestrin Glu 39, Glu 54, Glu 78, Glu 92, Asp 93 and Asp 101 and for the second calsequestrin Glu 199, Asp 245, Asp 278, Glu 350 and Glu 348.

The back-to-back form is stabilized by intermolecular interactions between the α4 helix of the domain II and the α3 helix of the domain I. The intermolecular salt bridges are between Glu 215 and Lys 86, Glu 216 and Lys 24, Glu 169 and Lys 85. There is also a hydrogen bond between Ala 82 and Asn 22. This dimerisation induces a very electronegative pocket at the C-terminal region which enables the binding of Ca2+.

Calcium Binding

Each monomere of calsequestrin can bind between 18 to 50 Ca2+. The Ca2+ ions bind to two or more acidic amino acids like Glutamate or Aspartate. These amino acids are mainly outside the calsequestrin or in the C-terminal region. When calsequestrins form homomeres, Ca2+ can bind in the electronegative pocket due to the front-to-front form and back-to-back form.

Ca2+ is not the only ion which can bind to the calssequestrin. One of them is Mg2+. The affinity is for Mg2+ is lower than the affinity for Ca2+ however the number of Ca2+ decrease. Another ion is H+. When the pH is low, more H+ will bind to the acidic amino acids and they can not bind Ca2+ anymore.

Regulation of CASQ2

There are 12 alpha helix and 15 beta sheet. The acidics amino acids can bind the Ca2+ especially the glutamate and the aspartate.

The Ct domain is highly implicated in the Ca2+ bounds. The Ca2+ is bound with the interaction of at least 2 acidic amino acids (glutamate or aspartate). These amino acids are in the external part of the protein. When Ca2+ binds to these amino acids there is a structural change which increase the number of alpha helix. Without Ca2+, there are 10-13% of alpha helix but in presence of Ca2+ there 20->35% of alpha helix. The N-term domain is implicated in front-to-front dimer interactions. While the C-term domain is involved in back-to-back dimer interactions.