Calmodulin

Importance of flexibility in calmodulin

Calmodulin binds such a wide variety of target proteins, making it especially important for it to have flexibility. Though Calmodulin's flexibility is more evident when it is bound to a target protein, NMR studies have shown that the linker region of Calmodulin is flexible, even when it is not bound to a target protein. Another important characteristic of calmodulin that allows it to bind a large variety of target proteins is the generic shape of the non-polar grooves in the binding sites. Since the non-polar grooves are generic, they don't require the target proteins to have any specific sequence of amino acids allowing a larger variety of target proteins to be bound. Together, these two structural characteristics of calmodulin allow it to flexibly bind target proteins with various shapes and amino acid sequences.^[5] For example, calmodulin binds both NMDA receptors and potassium channels which differ in length by about 50 amino acid residues.^[6][7]

On the left: Calmodulin with four calcium ions bound (1CLL.pdb). On the right: Troponin C with four calcium ions bound (1TCF.pdb).

Similarity to troponin C

Calmodulin's structure is very similar to the structure of troponin C (which is another calcium binding protein). They are both members of the EFh superfamily. Troponin C, like Calmodulin, has two globular domains that are connected by a linker region.^[8] However, Troponin C and Calmodulin differ in the length of the linker region; the linker region of Calmodulin is smaller than that of Troponin C.^[8] These remarkably similar structures are an example of how the EF hand motif is highly conserved in calcium binding proteins. Though they have similar structures, their functions are very different. Troponin C has a very specific function (to elicit a conformational change in Troponin I) ultimately causing a contraction in skeletal muscles. Calmodulin, evolved to bind a wider variety of target proteins, allowing it to play a role in many physiological events.^[5][8]

Overall

Solution structure of Calcium calmodulin C-terminal domain

Solution structure of Calcium-calmodulin N-terminal domain

Up to four calcium ions are bound by calmodulin via its four EF hand motifs.^[5] EF hands supply an electronegative environment for ion coordination. After calcium binding, hydrophobic methyl groups from methionine residues become exposed on the protein via conformational change. Using both X-Ray and NMR studies, scientists were able to determine that the conformational changes occurred in the alpha-helices of the EF motif, which changes the binding affinity for target proteins. When the alpha helices are perpendicular to one another, the Calmodulin is in an open conformation giving it a higher affinity for target proteins.^[9] More specifically, this conformational change presents hydrophobic surfaces, which can in turn bind to Basic Amphiphilic Helices (BAA helices) on the target protein. These helices contain complementary hydrophobic regions. The flexibility of calmodulin's hinged region allows the molecule to wrap around its target.^[5] This property allows it to tightly bind to a wide range of different target proteins. The C-domain of calmodulin has a higher affinity for calcium than does the N-domain.

Dynamic features

The C-terminal domain solution structure is similar to the X-ray crystal structure, while the EF hands of the N-terminal domain are considerably less open to the X-ray crystal structure. This indicates that Ca²⁺ binding causes a larger conformational change in the N-domain than in the C-domain. The backbone flexibility within calmodulin is key to its ability to bind a wide range of targets.^[10] Protein domains, connected by intrinsically disordered flexible linker domains, induce long-range allostery, or the conformational change of a protein by ligand binding to an allosteric site (a site other than the functional site), via protein domain dynamics.^[10]

General role in the body

Calmodulin mediates many crucial processes such as inflammation, metabolism, apoptosis, smooth muscle contraction, intracellular movement, short-term and long-term memory, and the immune response.^[11][12] Calcium participates in an intracellular signaling system by acting as a diffusible second messenger to the initial stimuli. It does this by binding various targets in the cell including a large number of enzymes, ion channels, aquaporins and other proteins.^[4] Calmodulin is expressed in many cell types and can have different subcellular locations, including the cytoplasm, within organelles, or associated with the plasma or organelle membranes, but it is always found intracellularly.^[12] Many of the proteins that Calmodulin binds are unable to bind calcium themselves, and use calmodulin as a calcium sensor and signal transducer. Calmodulin can also make use of the calcium stores in the endoplasmic reticulum, and the sarcoplasmic reticulum. Calmodulin can undergo post-translational modifications, such as phosphorylation, acetylation, methylation and proteolytic cleavage, each of which has potential to modulate its actions.

Specific examples

Role in smooth muscle contraction

Calmodulin bound to a peptide from MLC Kinase (2LV6.pdb)

Calmodulin plays an important role in excitation contraction (EC) coupling and the initiation of the cross-bridge cycling in smooth muscle, ultimately causing smooth muscle contraction.^[13] In order to activate contraction of smooth muscle, the head of the myosin light chain must be phosphorylated. This phosphorylation is done by Myosin Light Chain (MLC) Kinase. This MLC Kinase is activated by a calmodulin when it is bound by calcium, thus making smooth muscle contraction dependent on the presence of calcium, through the binding of calmodulin and activation of MLC kinase.^[13]

Another way that calmodulin affects muscle contraction is by controlling the movement of Ca²⁺ across both the cell and sarcoplasmic reticulum membranes. The Ca²⁺ channels, such as the ryanodine receptor of the sarcoplasmic reticulum, can be inhibited by calmodulin bound to calcium, thus affecting the overall levels of calcium in the cell.^[14] Calcium pumps take calcium out of the cytoplasm and/or store it in the endoplasmic reticulum and this control helps regulate many downstream processes.

This is a very important function of calmodulin because it indirectly plays a role in every physiological process that is affected by smooth muscle contraction such as digestion and contraction of arteries (which helps distribute blood and regulate blood pressure).^[15]

Role in short-term and long-term memory

Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in a type of synaptic plasticity called long-term potentiation (LTP) and requires the presence of calcium/calmodulin. CaMKII contributes to the phosphorylation of an AMPA receptor which increases the sensitivity of AMPA receptors.^[17] Furthermore, research shows that inhibiting CaMKII interferes with LTP.^[17]

Sorghum plant contains temperature-responsive genes. These genes help the plant adapt in extreme weather conditions such as hot and dry environments.

Role in plants

The plant sorghum is well established model organism and can adapt in hot and dry environments. For this reason, it is used as a model to study calmodulin's role in plants.^[18] Sorghum, contains seedlings that express a glycine-rich RNA-binding protein, SbGRBP. This particular protein can be modulated by using heat as a stressor. Its unique location in the cell nucleus and cytosol, demonstrates interaction with calmodulin that requires the use of Ca²⁺.^[19] By exposing the plant to versatile stress conditions, it can cause different proteins that enable the plant cells to tolerate environmental changes to become repressed. These modulated stress proteins are shown to interact with CaM. In an Arabidopsis thaliana study, hundreds of different proteins demonstrated the possibility to bind to CaM in plants.^[18] The CaMBP genes expressed in the sorghum are depicted as a “model crop” for researching the tolerance to heat and drought stress.