Calmodulin

Calmodulin is the quintessential member of the EF-hand family of calcium-binding proteins and functions as a key mediator in numerous signal transduction pathways. It is an acidic protein ) isoelectric point of 4.2) of molecular weight 16.8 kDa that is found in most eukaryotic cells, from yeast to humans. It is composed of two largely independent globular domains connected by a flexible central a-helix. The affinity of calmodulin for Ca²⁺ is fine-tuned to respond to intracellular calcium signals. Conformational changes within each of the domains induced by the binding of Ca²⁺ leads to the transduction of the Ca²⁺ signal.

1. Biological Function

Calmodulin is a signal transduction protein. It has been implicated in the control of a wide range of cellular functions, including cell proliferation, smooth muscle contraction, the regulation of ion channels, long-term potentiation and memory, and exocytosis. Furthermore, it has recently been found inside the nucleus, where it is thought to be involved in the regulation of DNA replication, gene expression, and DNA repair. In a resting cell with basal levels of Ca²⁺ , calmodulin exists in the inactive apo state. When a calcium signal is initiated and the intracellular levels of Ca²⁺ increase, calmodulin binds Ca²⁺ ions. This causes the protein to undergo a dramatic conformational change that exposes a large hydrophobic patch on the protein. This newly exposed hydrophobic surface then interacts with various cellular proteins, modulating their activity and thereby transducing the calcium signal.

 Protein kinases and phosphatases are among the best studied of calmodulin's targets. These enzymes are activated by the release of an autoinhibitory domain that is bound to the active site in the resting state. Ca²⁺-loaded calmodulin activates these proteins by binding to a site near to or overlapping with the autoinhibitory domain, causing the auto-inhibitory domain to dissociate from the active site. Myosin light chain kinase and calcium-calmodulin-dependent protein kinase II are two well-known examples of this class of calmodulin-regulated proteins. Calmodulin also regulates proteins involved in the generation of other second messengers, such as calmodulin-dependent cyclic nucleotide phosphodiesterase and nitric oxide synthase. The mechanism of regulation of these enzymes is thought to be very similar to that used to regulate the kinases and phosphatases. Calmodulin has also been shown to interact with proteins of the cytoskeleton and their regulatory proteins, such as spectrin and brush border myosin. However, the biological significance of these interactions is still unclear.

Recently, interactions between apocalmodulin and targets such as neuromodulin and unconventional myosins have been described. These interactions are thought to be mediated by an “IQ motif” (with the consensus sequence IQXXXRGXXXR, where X is any amino acid) in the target. This motif and its interaction with a Ca²⁺-free EF-hand calcium-binding protein was first described in myosin (1, 2). The functional significance of the interaction of proteins containing the IQ motif with apocalmodulin is not clear. It has been suggested, however, that perhaps neuromodulin, which is associated with the plasma membrane, functions as a trap for calmodulin, sequestering it near the membrane in the absence of the activating calcium signal (3).

 2. Ion-Binding Properties

 Calmodulin binds four calcium ions, with a dissociation constant of about 10^–6 M. Binding is selective for Ca²⁺; calmodulin does not bind Mg²⁺ or monovalent ions with appreciable affinity. The binding of Ca2+ is also highly cooperative, which is very important for calmodulin's biological function as a calcium sensor, because it allows for a tightly controlled “all or nothing” response to the calcium signal: the four binding events all occur within a very narrow range of Ca²⁺ concentration. If these binding events were spread over a large range of Ca²⁺ concentrations, calmodulin would be at least partially activated over the entire range. The system would therefore lack the required sharp separation between the activate and inactive states.

3. Structure

Calmodulin is a largely helical protein composed of four EF-hand motifs organized into two independent globular domains, each of which contain two EF-hands. The two domains are connected by a long, flexible central a-helix, giving the protein a dumbbell-like appearance (Figure 1a). This

quaternary structure is relatively similar in the apo- and Ca²⁺-loaded states of the protein. However, significant conformational changes occur within the individual domains on Ca²⁺ binding (Fig. 1b. In the apo state, each domain occupies the “closed” conformation (4-6). In this conformation, the four helices in the domain are nearly antiparallel, with interhelical angles near 180°. In Ca²⁺-loaded calmodulin, each domain occupies an “open” conformation in which the four a-helices are nearly perpendicular to each other (7, 8). Each domain exposes a hydrophobic surface of about 1.25 × 10^–8 m² in this open conformation (5). 

Figure 1. Ribbon representations of the three-dimensional conformations of (Ca²⁺)₄-calmodulin in the (a) absence and (b) presence of a target peptide. The peptide-free diagram was constructed using the coordinates of 1CLL (8). The two domains at the ends of the central a-helix consist of two EF-hand motifs. The diagram of (Ca²⁺)₄-calmodulin and the peptide analog of the myosin light chain kinase was constructed using the coordinates of 2BBM (10). The bound peptide is depicted darker than calmodulin.

4. Interactions with Target Peptides

The biophysical characterization of the interaction between calmodulin and its targets is often studied using small peptides (10–20 amino acid residues) derived from the calmodulin-binding omain of target enzymes. Calmodulin binds to these target peptides extremely tightly, with dissociation constants ranging from 10^–7 to 10^–11 M. The peptides adopt an amphiphilic helical conformation, and tend to have a bulky hydrophobic residue at either end, often (but not always(spaced 12 residues apart. However, the sequences of the target peptides are not highly similar. Calmodulin is able to bind so tightly to such a wide array of targets because of its own plasticity. The flexible central a-helix connecting its two domains can function as an “expansion joint,” allowing calmodulin to bind to peptides with different numbers of residues between the two bulky hydrophobic anchors. Furthermore, van der Waals interactions, which can be rather nonspecific, tend to dominate as the critical components stabilizing the interaction between calmodulin and the peptides. Hydrogen bonds, which are more structurally specific, are not as important in these interactions. The adaptability of the peptide binding surface of calmodulin may be further aided by its high proportion of methionine residues. Methionine is an unusually flexible and polarizable amino acid, which may allow calmodulin to mold its peptide-binding surface to meet the requirements of many different peptide sequences (9, 10).

High resolution three-dimensional structures of three complexes of calmodulin and peptides derived from target enzymes have been reported (11-13). These structures show that the relative disposition of the two domains of calmodulin is altered by the binding of a target peptide, but there is little change within the Ca²⁺-activated domains themselves. The calmodulin–peptide complex forms a well-packed ellipsoid, which contrasts sharply with the dumbbell shape of calmodulin observed in the absence of target (Fig. 1). The two domains of calmodulin essentially wrap around the target peptide, forming a hydrophobic tunnel in which the peptide binds. As of yet, there are no structures of calmodulin bound to an entire target protein. It is thought, however, that the mode of binding to the target sequence on the intact enzyme will be very similar to that seen in the calmodulin–peptide complexes.

References

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13. W. E. Meador, A. R. Means, and F. A. Quiocho (1993) Science 262, 1718–1721.