Calmodulin

Calmodulin is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells. It is an intracellular target of the secondary messenger Ca²⁺, and the binding of Ca²⁺ is required for the activation of calmodulin. Once bound to Ca²⁺, calmodulin acts as part of a calcium signal transduction pathway by modifying its interactions with various target proteins such as kinases or phosphatases.

Structure

Calmodulin is a small, highly conserved protein that is 148 amino acids long. The protein has two approximately symmetrical globular domains each containing a pair of EF hand motifs separated by a flexible linker region for a total of four Ca²⁺ binding sites, two in each globular domain. In the Ca²⁺-free state, the helices that form the four EF-hands are collapsed in a compact orientation, and the central linker is disordered; in the Ca²⁺-saturated state, the EF-hand helices adopt an open orientation roughly perpendicular to one another, and the central linker forms an extended alpha-helix in the crystal structure, but remains largely disordered in solution. The C-domain has a higher binding affinity for Ca²⁺ than the N-domain.
Calmodulin is structurally quite similar to troponin C, another Ca²⁺-binding protein containing four EF-hand motifs. However, troponin C contains an additional alpha-helix at its N-terminus, and is constitutively bound to its target, troponin I. It therefore does not exhibit the same diversity of target recognition as does calmodulin.

Importance of flexibility in calmodulin

Calmodulin's ability to recognize a tremendous range of target proteins is due in large part to its structural flexibility. In addition to the flexibility of the central linker domain, the N- and C-domains undergo open-closed conformational cycling in the Ca²⁺-bound state. Calmodulin also exhibits great structural variability, and undergoes considerable conformational fluctuations, when bound to targets. Moreover, the predominantly hydrophobic nature of binding between calmodulin and most of its targets allows for recognition of a broad range of target protein sequences. Together, these features allow calmodulin to recognize some 300 target proteins exhibiting a variety of CaM-binding sequence motifs.

Mechanism

Binding of Ca²⁺ by the EF-hands causes an opening of the N- and C-domains, which exposes hydrophobic target-binding surfaces. These surfaces interact with complementary nonpolar segments on target proteins, typically consisting of groups of bulky hydrophobic amino acids separated by 10–16 polar and/or basic amino acids. The flexible central domain of calmodulin allows the protein to wrap around its target, although alternate modes of binding are known. "Canonical" targets of calmodulin, such as myosin light-chain kinases and CaMKII, bind only to the Ca²⁺-bound protein, whereas some proteins, such as NaV channels and IQ-motif proteins, also bind to calmodulin in the absence of Ca²⁺. Binding of calmodulin induces conformational rearrangements in the target protein via "mutually induced fit", leading to changes in the target protein's function.
Calcium binding by calmodulin exhibits considerable cooperativity, making calmodulin an unusual example of a monomeric cooperative binding protein. Furthermore, target binding alters the binding affinity of calmodulin toward Ca²⁺ ions, which allows for complex allosteric interplay between Ca²⁺ and target binding interactions. This influence of target binding on Ca²⁺ affinity is believed to allow for Ca²⁺ activation of proteins that are constitutively bound to calmodulin, such as small-conductance Ca²⁺-activated potassium channels.
Although calmodulin principally operates as a Ca²⁺ binding protein, it also coordinates other metal ions. For example, in the presence of typical intracellular concentrations of Mg²⁺ and resting concentrations of Ca²⁺, calmodulin's Ca²⁺ binding sites are at least partially saturated by Mg²⁺. This Mg²⁺ is displaced by the higher concentrations of Ca²⁺ generated by signaling events. Similarly, Ca²⁺ may itself be displaced by other metal ions, such as the trivalent lanthanides, that associate with calmodulin's binding pockets even more strongly than Ca²⁺. Though such ions distort calmodulin's structure and are generally not physiologically relevant due to their scarcity in vivo, they have nonetheless seen wide scientific use as reporters of calmodulin structure and function.

Role in animals

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. 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. 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. 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 plays an important role in excitation contraction coupling and the initiation of the cross-bridge cycling in smooth muscle, ultimately causing smooth muscle contraction. 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 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.
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. Calcium pumps take calcium out of the cytoplasm 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.

Role in metabolism

Calmodulin plays an important role in the activation of phosphorylase kinase, which ultimately leads to glucose being cleaved from glycogen by glycogen phosphorylase.
Calmodulin also plays an important role in lipid metabolism by affecting calcitonin. Calcitonin is a polypeptide hormone that lowers blood Ca²⁺ levels and activates Gs protein cascades that leads to the generation of cAMP. The actions of calcitonin can be blocked by inhibiting the actions of calmodulin, suggesting that calmodulin plays a crucial role in the activation of calcitonin.

Role in short-term and long-term memory

plays a crucial role in a type of synaptic plasticity known as long-term potentiation which requires the presence of calcium/calmodulin. CaMKII contributes to the phosphorylation of an AMPA receptor which increases the sensitivity of AMPA receptors. Furthermore, research shows that inhibiting CaMKII interferes with LTP.

Role in plants

While yeasts have only a single CaM gene, plants and vertebrates contain an evolutionarily conserved form of CaM genes. The difference between plants and animals in Ca²⁺ signaling is that the plants contain an extended family of the CaM in addition to the evolutionarily conserved form. Calmodulins play an essential role in plant development and adaptation to environmental stimuli.
Calcium plays a key role in the structural integrity of the cell wall and the membrane system of the cell. However, high calcium levels can be toxic to a plant's cellular energy metabolism and, hence, the Ca²⁺ concentration in the cytosol is maintained at a submicromolar level by removing the cytosolic Ca²⁺ to either the apoplast or the lumen of the intracellular organelles. Ca²⁺ pulses created due to increased influx and efflux act as cellular signals in response to external stimuli such as hormones, light, gravity, abiotic stress factors and also interactions with pathogens.

CMLs (CaM-related proteins)

Plants contain CaM-related proteins apart from the typical CaM proteins. The CMLs have about 15% amino acid similarity with the typical CaMs. Arabidopsis thaliana contains about 50 different CML genes, which leads to the question of what purpose these diverse ranges of proteins serve in the cellular function. All plant species exhibit this diversity in the CML genes. The different CaMs and CMLs differ in their affinity to bind and activate the CaM-regulated enzymes in vivo. The CaM or CMLs are also found to be located in different organelle compartments.

Plant growth and development

In Arabidopsis, the protein DWF1 plays an enzymatic role in the biosynthesis of brassinosteroids, steroid hormones in plants that are required for growth. An interaction occurs between CaM and DWF1, and DWF1 being unable to bind CaM is unable to produce a regular growth phenotype in plants. Hence, CaM is essential for the DWF1 function in plant growth.
CaM binding proteins are also known to regulate reproductive development in plants. For instance, the CaM-binding protein kinase in tobacco acts as a negative regulator of flowering. However, these CaM-binding protein kinase are also present in the shoot apical meristem of tobacco and a high concentration of these kinases in the meristem causes a delayed transition to flowering in the plant.
S-locus receptor kinase is another protein kinase that interacts with CaM. SRK is involved in the self-incompatibility responses involved in pollen-pistil interactions in Brassica.
CaM targets in Arabidopsis are also involved in pollen development and fertilization. Ca²⁺ transporters are essential for pollen tube growth. Hence, a constant Ca²⁺ gradient is maintained at the apex of pollen tube for elongation during the process of fertilization. Similarly, CaM is also essential at the pollen tube apex, where its primarily role involves the guidance of the pollen tube growth.