The Calcium bowl

The calcium bowl is presumed to be found within the DQDDDDDPD sequence [see fig. 7]  because point mutations in this sequence resulted in the CTD being less sensitive to to calcium. This was also proven in other studies which showed good binding of calcium to the region of interest by using a radioactive calcium assay and activation of the channel.

The shown interactions (yellow dotted line[see fig. 9]) are between the calcium ions and Q889, D892, D895 and D897. Through the use of alanine-scanning mutagenesis, it was discovered that of the aforementioned amino acids D895 and D897 are the most important residues for Ca²⁺  sensing.

Fig. 9: Stick figure representations of the calcium bowl in BK channel (left) and the calcium binding region in the non-lysosomal calcium activated cysteine protease, calpain. We can appreciate the similar geometrical structure of the calcium ions and their respective binding sites in both these proteins.

Further evidence of the interaction between the Ca²⁺ bowl and the Ca²⁺ ion were elucidated through this x-ray crystal. This allowed us to take a look at the conformation of the surrounding amino acids through the increased resolution of the 3.0 Å image that allowed the authors to place the side chains in the, what they presume, is the correct orientation. This allowed the authors to hypothesize that the Ca²⁺ bowl is found between βO and βP (last two beta strands of the RCK2 Rossman fold [see fig. 7]) in the tertiary structure.  Also, it has been found that the atoms in direct contact with the ion are the carbonyl oxygens of Q889 and D892 as well as the oxygen atom from the D895 and D897 side-chains. A similar geometrical structure has been found in a cysteine protease which is regulated by Ca²⁺ called calpain.

While the electron density maps allow for some discrepancy with the postulated model, mutagenic studies have supported this model. The two most important residues for protein function are D895 and D897 which correlates with the idea of these two residues being involved in the co-ordination of the calcium ion. The third most important residue is D894 which is thought to stabilize the structure of the calcium bowl by forming salt bridge interactions with R1018 and K1030. 

Fig. 10: Figure highlighting the salt bridge interaction between residue D894 and R1018 and K1030 that stabilizes the structure of the calcium bowl. Please click to enlarge.

However, D896 mutation showed to have no effect on the function of the protein. This can be explained by the fact that this residue's side chain is pointing away from the calcium and therefore plays no role in any important interaction.

Video 1: A journey through the CTD of the Human BK Channel highlighting key structural components such as the flexible interface and the calcium bowl, created using PyMol.