Calmodulin Calcium Binding Protein Complexed with TFP

Calmodulin, calcium modulatory protein, is a calcium binding protein that is located mainly in the cytosol and seems to regulate many calcium ion mediated events, like muscle contraction. Calmodulin also plays various roles in signal transduction pathways where it activates protein kinases. This protein binds four calcium ions and is highly conserved. Calmodulin contains, on average, 148 amino acids and has a molecular weight between 16.063 KD and 18.165 KD depending on the species from which it is isolated. Calmodulin is present in all eukaryotic cells and some plant cells.

When Calmodulin binds calcium hydrophobic sites are opened. This allows the protein to interact with other proteins; one example of this is the activation of calmodulin dependent kinase. The figure below shows calmodulin with four calcium atoms bound. Because calcium is bound to this protein the hydrophobic sites are open and the protein is ready to interact with other proteins in the vicinity. In order to see calcium bound to calmodulin click the button below

Calmodulin without bound TFP

This picture shows Calmodulin in water, the oxygen atoms are show to represent the solvent. The bound calcium is shown in blue. Other atoms present are chloride, red and sodium ion, grey.

A closer view of the interaction between calmodulin, calcium and water is seen in the next still image. This figure shows which amino acids in calmodulin interact with one clacium ion in aqueous solution. From the figure it is easy to see that the calcium ion interacts with Asp 24, 22 and 20, Thr 36, and Glu 31 as well as one water molecule. The bulk of the amino acids interacting with the calcium ion are negatively charged at physiological pH. This makes good sense since calcium has a positive two charge which must be stabilized by the protein.

This picture is a close up view of the interaction of water molecules and the calcium ion.

The binding site of each EF hand binds to calcium by six oxygens from the protein and one oxygen from a bound water molecule (as seen in the above figure). When calcium binds to calmodulin the protein is able to fold changing from a dumbell shape to a globular shape. The two lobes come together due to the flexible nature of the long alpha helix connecting the ends. Many of the residues in calmodulin are hydrophobic and are located next to negatively charged regions. The conformational changes caused by the binding of calcium means that these regions are exposed. This enables calmodulin to bind to the positively charged amphipathic regions of targets such as peptides composed of leucine, lysine and tryptophan or inhibitors like TFP. These bind very strongly to the calcium-calmodulin complex but not to free calmodulin. When the calcium-calmodulin complex binds to a target the target is enclosed in a hydrophobic pocket. The figure below shows the hydrophobic regions of calmodulin. As stated above it is these regions that interact with other proteins to the "business" of the protein.

This figure show the hydrophobic regions of calmodulin.

Next is a figure showing where sodium ions interact with calmodulin. This figure was created to show the negative nature of certain parts of the protein surface. The negative regions are necessary for calcium binding.

Left: Three-dimensional histogram of sodium ion distribution in calmodulin's vicinity sampled from the 3 ns trajectory. The colors code for the ion density values 1.0 M (red) and 3.0 M (yellow). Right: Contours of constant sodium concentration and electrostatic potential near the surface of the negatively charged protein. The contour was calculated with the program Grasp at 75 mM ionic strength. The colors code for the ion density values 0.1 M (blue) and 0.2 M (green), and 0.5 M (yellow).

The pictures following give a few different views of calmodulin without calcium bound. They show the cylinders present in the protein as well as the seven alph helices which contribute to the dumbell shape of the protein.

Still image of Calmodulin with cylinders shown

Still image showing the seven helices of calmodulin

The above pictures lack bound TFP - trifluoperazine. TFP is a small molecule that is an inhibitor of calmodulin. TFP binds to the hydrophobic regions of the protein preventing interactions with target proteins and thus preventing calmodulin from doing its job! Push the opening settings button above to see one view of calmodulin boud to TFP.

Another view of calmodulin bound to TFP is shown in the still image below. In this figure one can see that calmodulin is bent over so both hydrophobic sites are interacting with one TFP molecule. This globular shape, spoken of above, is probably more like what would be seen when calmodulin binds to a target protein in the cell.

Lastly, below is an image showing one of the binding sites of TFP with calmodulin. This figure was obtained from insight II. The active site is labeled in green and this is just one of the active sites for TFP on this protein. There is another similar site on the opposite end of the molecule.

In order to see different views of the calmodulin protein click the appropriate buttons on the right.

References:

Alberts, Bruce et al; Molecular Biology of the Cell, 2nd Edition; p.710-711; Garland Publishing Inc. New York, 1989

http://www.bio.nagaokaut.ac.jp/~senda/CaMabst.html

http://www.ks.uiuc.edu/Research/cell_motility/calmodulin/

http://www.physiology.cup.cam.ac.uk/Pr...ts/523P/Brimingham/Files/S227.html

http://structbio.vanderbilt.edu/cabp_database/general/prot_pages/calmod.html#chemphys