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Calmodulin is a Ca2+ binding protein that plays a key role in calcium signaling in eukaryotic cells. It can bind to and regulate the activity of various enzymes, ion channels, and other proteins involved in diverse cellular functions, such as metabolism, muscle contraction, gene expression, and cell survival-all-in-all it interacts with several hundred different proteins to control their activity. [1] It is a small (148 amino acid chain, 16.7 kDa) highly conserved protein that has four Ca2+ binding sites. Upon binding calcium cation, Ca2+-free CaM (apoCaM) undergoes significant conformational change. The Ca2+-bound calmodulin (holoCaM) transmits the signals and regulates other proteins by binding to calmodulin binding domains of the targeted proteins.
The structure in the left frame is that of apo-calmodulin (or Ca2+-free calmodulin) determined using solution NMR methods.
The tertiary structure can be divided in two domains: N-domain (blue, contains N-terminal) and C-domain (red, contains C-terminal) connected via a loop tether (white). Both domains are compact and globular in structure and the tether allows flexible movement of the two domains
Video 1.
The secondary structure of both domains is alpha-only except for two very short (three amino acids in length) beta strands (yellow) located in N-domain.
Each domain has four alpha helices labeled A through H starting from N-terminal.
In absence of structural Ca2+ cations, the hydrophobic amino acid residues in each domain to collapse and form a hydrophobic core inside the domain's globular structure. This is illustrated for N-domain here, with hydrophobic amino acids shown in white and their residues in stick model. The importance of these residues will be discussed below.
As the video below illustrates, the surface of apoCaM is mostly negatively charged.
Video 2. The surface potential of apoCaM.
The structure of Ca2+ loaded, holo calmodulin (holoCaM) is shown on the right. The color scheme is the same as for apoCaM. Perhaps the most noticeable difference between apo and holoCaM is in the structure of the tether (white). Here, the tether has a well-defined helical secondary structure. This, however, is likely the consequence of the structural method used for analysis of holoCaM–single crystal X-ray crystallography. It is probable that crystallization and crystal packing resulted in the formation of helix because, as we shall see later, this segment remains flexible in holoCaM.
Both domains bind two Ca2+ cations (green spheres).
Each cation binds to a loop flanked by two helices. This Ca2+ binding motif, the most common among Ca2+ binding proteins, is called EF hand. The name comes from the Ca binding site in parvalbumin (another small Ca2+ binding protein involved in signaling) located on a loop flanked by helices E and F. Almost perpendicular orientation of these two helices and the connecting loop resemble a hand with extended thumb and index fingers: the helices aling with fingers and bent middle finger corresponds to the loop. (Figure 1)
Figure 1. The EF hand motif for calcium ion binding (click on the thumbnail).
The EF hand motifs always come in pairs, never isolated. There is a solid evidence suggesting that they work in synergy and increase each other's affinity for the cation–an isolated EF-hand has 100 to 10 000 lower affinity for Ca2+ in comparison to the one in a pair. [2]
Each Ca2+ is in the same coordination environment. The donor atoms are exclusively oxygen atoms. These can be provided by carbonyl groups from either protein backbone (as is the case of Leu99 in the display on the left) or from asparagine's side chain C=O group (Asn97). More comon are negatively charged carboxyl groups from amino acids side chains. These can act as monodentate (Asp95 and Asp93) or chelating (Glu104) ligands. And finally, one (as is for this Ca2+) or two water molecules complete the coordination sphere.
The coordination number for all ions is seven resulting in . The overal charge on this Ca site is 1- (3- from three carboxylates and 2+ for one Ca cation).
Hydrogen bonds (dashed yellow lines) further stabilize the metal site and EF hand folding.
Figure 2 shows coordination of all four Ca2+ ions in holoCaM structure.
Figure 2. The coordination environments of calcium cations in holoCaM (click on the thumbnails).
The Ca2+ binding induces significant conformational changes in both N and C domain. Outward movement of helices opens up the globular structure and Figure 3 compares the conformations of N and C domains of apo and holoCaM, while Figure 4 illustrates the hydrophobic cavities formed in both domains.
Figure 3. Comparison of apo and holo calmodulin topologies.
Figure 4. Surface potentials of N and C domains in holoCaM looking at the hydrophobic cavities.
The surface potential is still predominantly negative (Video 3).
Video 3. The surface potential of holoCaM.
Each protein regulated by holoCaM has a calmodulin binding domain (CBD). The CBDs are segments, usually very short, to which holoCaM binds and transmits the signal. One such domain, from plasma membrane Ca2+-ATPase (PMCA) pump is shown on the left. It is 28 amino acids in length, and it forms an alpha helix. To put the size of the CBD in perspective, the primary sequence of PMCA contains 1220 amino acids, meaning that CBD comprises about 2.2% of the structure.
The two hydrophobic amino acids, Trp1093 and Phe1110 are essential for the recognition of and serve as anchors for holoCaM.
The holoCaM attaches to these residues using the hydrophobic cavities in both lobes and wraps around the CBD in an antiparallel fashion: N-domain of holoCaM is attached to the C-end of the CBD. The middle tether has no secondary structure which allows holoCaM to colapse around the CBD.
A closer look at the interactions between hydrophobic region of holoCaM's N domain and Phe1110 on CBD.
The hydrogen bonds along the tether ensure proper twist around the CBD.