CaM is a small (17 kDa) Ca2+-binding protein with ubiquitous express in eukaryotic cells. It is one of the famous Ca2+ sensor and adaptor protein, which contributes tremendously in understanding about processing of Ca2+ signals by EF-hand proteins. The crystal structure of parvalbumin is a historical landmark in Ca2+ signaling leading to the EF-hand-protein concept. Through the interaction with numerous targets, CaM transfers Ca2+ signals to regulate multitudes of intracellular processes such as cell growth, proliferation, motility, and apoptosis. The concept of EF-hand was first devised in 1973 when the parvalbumin crystal structure was solved (24). The canonical EF-hand in CaM is characterized by a 29-residue helix-loop-helix motif as shown in figure 1.2. Ca2+ in the EF-hand is coordinated by oxygens from sidechain carboxyl of amino acids at position 1, 3, 5 and 12, from main chain carboxyl of amino acid at position 7, and a bridged water at position 9. The topology of EF-hand resembles the spread thumb and forefinger of our hand. The EF-hands in two globular domains of CaM display different binding affinities for metal ions. The Ca2+-binding affinity of C domain is 3-fold higher than that of N domain. Four sites from two globular domains function cooperatively to bind Ca2+. In the absence of Ca2+, CaM is in a “closed conformation” with antiparallel EF-hand helices from two domains. Upon Ca2+-binding, CaM adapts to an “open conformation” in which helices are more perpendicular to each other, exposing more hydrophobic surface for a wide range of targets binding (25) .
Figure 1.2 EF hand (26)
(a) Cartoon representation of EF hand. The canonical EF hand is formed by helix-loop-helix. Ca2+ ion in the EF hand is coordinated by ligands inside of this 12-residue loop region.
Oxygen coordinating Ca2+ ions are from side chain of amino acids at position 1, 3, 5 and 12,
main chain of amino acid at position 7, and water at position 9. (b) Crystal structure of EF in CaM.
Recent work involving crystallization, nuclear magnetic resonance (NMR), and mutagenesis studies have revealed how CaM interact with and regulate its targets in various different ways. Typically, before binding with its targets reversibly or irreversibly, CaM needs to be activated by Ca2+. However, apo-CaM can also bind to the targets in many cases. CaM-binding
proteins possess specific regions characterized with a net positive charge, moderate hydrophilicity, and moderate to high helical hydrophobic moment (27,28). Many target sequence are intrinsically disordered and undergo a disorder-to-order conformational transition as reported (29-31). Those CaM-binding motifs are generally divided into IQ motif which is Ca2+-independent CaM binding motif and Ca2+-dependent CaM binding motifs. Ca2+-dependent motifs can be further grouped based on the distance between hydrophobic anchor residues (28,30,32-36).
IQ motif represents a large group of CaM-binding sequences. One of the well-known characteristics of IQ motif is its Ca2+-independence in CaM binding. However, recently, more and more work found that some IQ motifs also bind with Ca2+-loaded CaM (37,38). Studies of CaM interaction with unconventional myosin provide primary elucidation of characteristics of IQ motif. IQ refers to the first two amino acids isoleucine and glutamine in the consensus sequence (IQXXXRGXXXR) of IQ motifs. The first residue isoleucine can be replaced by other hydrophobic amino acids such as leucine and valine. Glycine at position 7 is also ambiguous in some CaM-binding proteins like PEP19 and some myosin protein (39). Position 11 can be either arginine or lysine. A more generalized IQ motif can be presented as [I, L, V]QxxxRxxx[R, K]. Currently more IQ motif-containing proteins have been reported. Some of them called IQ-like motifs ([FILV]Qxxx[RK]xxxxxxxx) which does not strictly follow the generalized IQ motif sequence listed above. They are incomplete IQ motifs with only the first half of the IQ motif. Studies of the essential and regulatory light chain of conventional myosin revealed that C-domain of apo-CaM interacts with the first half of IQ motif (IQxxxR), while N-domain of apo-CaM binds with the second half without inducing significant conformation change of N-domain. The binding affinity of IQ motifs to CaM varies among different IQ motifs and Ca2+ concentrations.
How CaM interacts with its targets and regulates its binding partners varies. Through analyzing the CaM complex structures deposited in protein data bank (PDB), CaM-binding could be grouped into two general categories: extended and collapsed (Fig. 1.3). In extended mode, two domains of CaM interacts with different site of targets. The distance between two domains of CaM is not significantly influenced. The collapsed mode is also called canonical wrap-around mode, in which two domains of CaM bind to the hydrophobic anchor residues in the binding motif and form a hydrophobic pocket which wraps the helical target inside. The distance between two domains could be largely shortened by the flexible linker from 50 Å to less than 10 Å. Figure 1.4 shows that CaM regulates various cytosolic and membrane proteins including gap junctions. How Ca2+/CaM regulates connexins especially Cx45 for cell-cell communication will be addressed in Chapter 3.
Figure 1.3 CaM function as an adaptor protein to interact with various target proteins.
Ca2+-free and Ca2+-load CaM are able to bind with a wide range of targets including soluble
cytosolic proteins and membrane proteins.
Figure 1.4 Diverse CaM-binding modes.
CaM interact with various target proteins in different binding modes. These modes can be divided into two groups-collapsed and extended, based on whether the distance between N-