Please be patient while the structures in the left frame load. In order to display all of the structures in the tour properly, press 'View' buttons below in order (from 1 to the end).
Calsequestrin (CASQ) is a primary Ca2+ storage and buffering protein fond in sarcoplasmic reticulum (SR, a membrane-bound organelle responsible for Ca2+ homeostasis) where it controls the concentration of free, uncomplexed Ca2+ cation and regulates its influx. Calsequestrin's high capacity for Ca2+, about 40-50 mol of cation per mol of CASQ, ensures low concentration of free Ca2+ in SR. However, its low Ca2+ binding affinity (Kd) permits easy dissociation of the complex when Ca2+ is required for action. [1], [2]
There are two CASQ isoforms: CASQ1, found in skeletal muscle and CASQ2, found in hart muscle. Both are relatively small proteins with about 400 amino acids in primary sequence (for human CASQs) and molecular mass of ≈45 kDa. The two isoforms share high primary structure identity (60-70%, [3]) and overall structural similarity but CASQ2 has two highly conserved cysteine residues. [2]
The Ca2+-free, apo-CASQ, does not have a well-defined secondary or tertiary structure. [4] As the concentration of Ca2+ increases, the protein folding starts and at Ca2+ concentrations of about 0.2 mM the folded, α/β CASQ structure is established. Such folded structure of holo-CASQ1 from human skeletal muscle is shown on the left in ball-and-stick model. Once the Ca2+ concentration reaches ≈0.3 mM, CASQ starts forming dimers. With further increase in the cation concentration CASQ1 gives tetramers (dimers of dimers) and later octamers. The polymerization significantly increases CASQ1 Ca2+ binding and buffering capacity (by some accounts for about 40%). Unlike CASQ1, CASQ2 remains mostly in monomeric and dimeric form. [2]
The structure of holo-CASQ1 consists of three domains, each sown in different colour (Domain I, red; Domain II, green and Domain III blue).
All three domains have nearly identical tertiary structure with a β-sheet composed of four β-strands flanked by two pairs of α-helices on both sides. This topology corresponds to a thioredoxin fold; a very unusual one for Ca-binding proteins (Figure 1). [5] Thioredoxins are small, redox proteins but this fold occurs in about 4000 proteins with diverse functions. [2]
Figure 1. Thioredoxin fold in CASQ (click on the thumbnail).
The fold places hydrophobic amino acid residues in the core of the fold (white regions in the display) while acidic residues (red) face the exterior. The primary sequence of CASQ contains about 30% of aspartic and glutamic acid, both of which are deprotonated under physiological pH, and significant percentage of protein's exterior has negative charge density (Figure 2).
Figure 2. The primary sequence and the potential surface map of human CASQ1 (click on the thumbnails).
This view shows the distribution of aspartates (red) and glutamates (teal) in the tertiary structure.
Both CASQs isoforms have two types of Ca2+ binding sites—the high and low affinity.
There are only three high affinity sites for Ca2+ biding (green spheres). They are considered as such because most of the donor atoms for Ca2+ come from the protein, they are buried deeper inside the structure (in comparison to low affinity sites) and are believed to significantly contribute to CASQ folding. The high affinity sites are not equally distributed among domains–domain I has one such site, domain II has two, but domain III has none.
One such site is shown on the left. The Ca2+ cation is coordinated by three protein residues Asn17, Val18 and chelating aspartate. The coordination is completed by one water molecule to give a distorted geometry.
Hydrogen bonding also stabilizes the coordination as is the case here with coordinated water molecule interacting with a leucine and valine residues (yellow dashed lines).
The details of Ca2+ coordination at other two high affinity sites are shown on Figure 3.
Figure 3. Two high affinity binding sites in human CASQ1 (click on the thumbnails).
In contrast, in low affinity sites Ca2+ is on the surface of the protein and is coordinated mostly by water molecules. The example onthe left shows a Ca2+ in one such site coordinated by five water molecules and a chelating thyrosine giving a distorted pentagonal bipyramidal and coordination number seven. The structure in this tour has 12 Ca2+ cations located in low affinity sites.
The first step in CASQ1 polymerization is formation of dimers. One such is shown on the right with monomers shown in white and light blue.
The N-terminus of each monomer docks into mostly hydrophobic cavity located between two helices of another monomer. Figure 4 shows this interaction using surface display of one of the monomers.
Figure 4. The N terminus interactions (click on the thumbnails).
This interaction brings acidic residues of two monomers in close proximity creating a long channel, capable of hosting more Ca2+ ions in the low affinity sites (Figure 5).
Figure 5. The negatively charged channel on the interface between two monomers (click on the thumbnail).
Video. CASQ1 dimer fly-by.
As the concentration of Ca2+ increases, the dimers further dimerize, this time in back-to-back fashion through C-termini, to create tetramers. One such tetramer is shown now on the left.
The back to back interaction involves Asp and Glu residues in Domain III of one dimer (C-terminus) and in domains I and II of the other dimer. These are held together with Ca2+, two of which are shown on the display, and hydrogen bonding (dashed red lines). The cation's coordination environment is completed with water molecules.
The oligomerization of CASQ1 continues to at least octameric, linear state called CASQ filament. Figure 6 shows a structure of a hexameric filament (from PDB ID 6OWV. In vivo imaging, however, suggests the existence of higher, as well as branched, oligomers of CASQ1.
Figure 6. A short, hexameric filament of CASQ1 (click on the thumbnails).
CASQ controls the intake of Ca2+ ions to the muscle cells by biding to skeletal muscle calcium release channel (Ryanodine receptor 1 or RyR1) in the intermediate Ca2+ concentration range, ≈1 mM. Under this concentration regime, CASQ1 is a tetramer (a dimer of dimers). The binding of CASQ1 to RyR1 inhibits the channel. Any other form of CASQ1 (monomer, dimer of filament) dissociates from RyR1 and activates the channel.
This is the structure of human CASQ1 monomer. Fell free to play with it.