Created by Xiang Li

   Calcium binding proteins regulate many cellular processes. There are two groups of calcium binding proteins, and calmodulin (1CLL) belongs to the group of calcium-modulated proteins, which all have EF hand as a structural feature. Calmodulin modulates the activity of other proteins such as protein kinase, NAD kinase, phosphodiesterase, and calcium pumps (3). Calmodulin mediates the control of a large number of enzymes, ion channels, and other proteins by calcium. Together with CEP110 and centrin, calmodulin is also involved in the pathway that regulates the centrosome cycle and progression through cytokinesis (7).

   Calmodulin has 148 amino acid residues. Its molecular weight is 16707.39 Da, and its isoelectric point (pI) is 4.09. The source of calmodulin is Homo sapiens. Calmodulin is dumbbell-shaped. It has two globular domains connected by a central alpha-helix. Each domain has three alpha-helices and two calcium binding EF hands. The EF domains have a degree of sequence homology as low as 25%, but their three-dimensional structures are nearly identical (1). There is a short antiparallel beta-sheet between adjacent EF hands and one non-EF hand. The two beta-sheets are held together by two hydrogen bonds each. The secondary structure of calmodulin includes alpha-helices, beta-sheets, and random coils. Two thirds of the 148 residues are in alpha-helical conformation. The seven alpha-helices comprising residues 5 to 19 (I), 29 to 37 (II), 45 to 55 (III), 65 to 92 (IV), 102 to 111 (V), 118 to 128 (VI), and 138 to 146 (VII) consist mainly of polar and charged residues (2).

   Calmodulin has only one subunit. It contains no prosthetic groups. The crystallization of calmodulin has not been done without including calcium. The ligands of calmodulin are calcium ion and ethanol. Calcium acts as a sensor and signal transducer. Changes in intracellular calcium concentration regulate calmodulin by directing its subcellular distribution, promoting different types of association with target proteins, and resulting in various conformations of calmodulin that activate target-specific proteins (4). Ethanol inhibits calmodulin binding to synaptic plasma membrane, which correlates with the increase of membrane fluidity (5). The ethanol molecule explains the crescent-shaped density inside the amino-terminal hydrophobic pocket when ethanol is used for crystallization (2).

   When calmodulin binds to calcium, its functional configuration changes dramatically and enables calmodulin to bind to various protein targets with extremely high affinities. All calmodulin target proteins have a basic amphiphilic alpha helix (a Baa helix) which calmodulin binds to specifically. A Baa helix has hydrophobic residues on one face and basic residues are opposite face. The Baa helices exhibit extreme variability in sequence, while calmodulin itself is highly conserved. Each globular domain of calmodulin has a large hydrophobic surface with highly negative electrostatic potential, which is suitable for interactions with a Baa helix. When calmodulin binds to the target protein, its two globular domains fold together. The long central alpha-helix (residues 65-92) serves as a flexible tether that allows the two globular domains to adjust their orientation for maximal binding of the target protein (1).

   Within the calmodulin molecule, there is only one short hydrogen bond of 2.1 Å between Glu-82 and Arg-86 side-chains. Other hydrogen bonds shorter than 2.5 Å involve water molecules as acceptors. All the residues have their main-chain amide involving in hydrogen bonding with acceptors except for eight residues for which the hydrogen bonds are donated to water molecules and Glu-45 which has not partner closer than 3.5 Å. Asp-118, a surface polar side-chain, has double conformation. One of the conformations makes hydrogen bonds to the side-chains of Arg-106 directly, and the other makes hydrogen bonds to Arg-106 via a water molecule (2).

   In the intervening region between two alpha-helical regions from residue 38 to 45, there is a hairpin turn made possible by Gly-40. There are two other turn in calmodulin that are not EF hand turns, consist of residues 39 to 44 (alpha helices II and III) and residues 112 to 117 (alpha-helices V and VI). Both turns have a Gly residue. The turns are stabilized by three hydrogen bonds each (2).

   Met is one of the critical residues of calmodulin. Calmodulin contains nine Met residues out of its 148 amino acids, which is much higher Met content than other proteins. Four Met residues are clustered in each of the globular domains at residues 36, 51, 71, 72, 109, 124, 144, and 145. The ninth Met residue is located at position 76 in the linker region. The highly localized distribution of Met residues accounts for approximately half the surface area of the hydrophobic target-binding interface of calcium and calmodulin. Met residues in calcium-calmodulin complex are susceptible to oxidation. Once oxidized, the polar Met sulfoxide (MetO) will reduce the efficacy of target regulation by calmodulin. Met residues in calmodulin have been highly conserved by evolution (10).

   Furthermore, the amino acids in the linker region, residues 74 through 80, also contribute to the stability and calcium affinity of calmodulin. Residues Lys-75 and Asp-80 in the region are completely conserved among many species; which implies that they are most critical for calmodulin function. This region of calmodulin consists of polar and charged residues only. Residues 74 to 77 add to the relative proportion of positively charge sidechains. Interactions between either Arg-74 or Lys-75 and Glu-78 contribute to the stabilization of helix IV in the N-domain fragment. The contacts that Lys-75, Met-76, and Lys-77 make with distal residues in helices I, II, and III are critical in defining the structural stability and calcium affinity of the fragment. Residues 78 through 80 have an alternative role: contacts made between these residues and C-terminal residues 81 through 84 contribute to the overall mechanism in which signals from the C-domain are propagated to the N-domain (11).

   Blast search results show that the EFh superfamily is the conserved domain for calmodulin and other similarly aligned proteins. This result agrees with the function of calmodulin; because EF hand is the calcium binding motif, and the EFh superfamily include a variety of calcium sensors and calcium signal modulators. The calcium binding sites are also conserved (6).

   One of the proteins that are similar to calmodulin is troponin C (1TCF). The Blast sequence alignment results in an E value of 9e-50, and an identities percentage of 54%. The Z score from Dali search for troponin C is 14.8. These show that troponin C is similar to calmodulin both in amino acid sequence and in three-dimensional structure. Troponin C is a calcium-binding regulatory protein in skeletal and cardiac muscles. It regulates the interaction between myosin crossbridges and actin in response to intracellular calcium concentration. Troponin C is a calcium-modulated protein like calmodulin. They both belong to the EFh superfamily. The source of troponin C is Oryctolagus cuniculus (rabbit) (8).

   The molecular weight of troponin C is 17974.84 Da, and its isoelectric point (pI) is 4.06. The pI of calmodulin is 4.09, so the two proteins have approximately the same pI. Troponin C has 159 amino acid residues, which is longer than calmodulin; hence troponin C is about 1200 Da heavier than calmodulin. The ligand of troponin C is also calcium ion. Troponin C contains four EF hands, therefore four calcium-binding sites. Two of them are high-affinity sites in the C-domain that also bind magnesium and are always occupied. The other two are low affinity calcium-specific sites in the N-domain (8). Where as in calmodulin, all the calcium-binding sites bind with high affinities.

   Calmodulin has evolved to bind many target proteins and regulate the function of a variety of enzymes. Although they are both calcium-modulated proteins, troponin C is a bistable switch that controls contraction in striated muscles and it has a specific target, troponin I. Troponin I is the inhibitory subunit of the troponin complex that is on the thin filaments of muscle. When troponin C binds to 4 calcium ions, it undergoes a conformational change which is then transmitted to troponin I, making troponin I release its inhibition of the actin-myosin interaction via troponin T and tropomyosin (9).

   Calmodulin and troponin C are the most similar members within the EFh superfamily. Mutation of troponin C that mimics calmodulin is used to examine the factors that determine their specificity as regulatory proteins. A double mutation of troponin C that resembles calmodulin in lacking both the N-terminal helix and residues 91 to 93 shows a small difference from troponin C in binding to the erythrocyte Ca2+-ATPase, and an improvement in enzyme activation. In addition, residues 88 to 90 are replaced with corresponding sequence from calmodulin as a triple mutation of troponin C. This result is equivalent to calmodulin in maximal activation, and it restores protein ability to increase calcium affinity for enzyme. However, the triple mutant binds less tightly than calmodulin. Furthermore, temperature decrease has a more significant effect in protein binding than either mutation, decreasing the difference in affinities to 18-fold, but with no improvement in the ability to increase calcium affinity for the enzyme (12).

   From Blast and Dali search results, some other proteins that are similar to calmodulin are: myosin VI (2VB6) from Sus scrofa (pig) and Gallus gallus (chicken) with an E value of 1e-86 and a Z score of 13.2; calcium bound dimeric GCaMP2 (3EVU) from Aequorea victoria (jellyfish) with an E value of 7e-99 and a Z score of 14.3; and skeletal muscle troponin T from Gallus gallus (chicken) with an E value of 3e-49 and a Z score of 13.9.

   ProSite search of calmodulin gives a functional motif of sequence EQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQN. The top three structures with threedimensional structure alignment to calmodulin obtained from VAST are compared using Cn3D. The Prosite functional motif is almost completely conserved in double mutant Q41LK75I of N-domain of calmodulin from Homo sapiens (2I08), which has an E value of 1e-47 and a Z score of 12.9. The other two top structures with three-dimensional alignment to calmodulin are centrin-XPC peptide from Homo sapiens (2OBH) with an E value of 4e-45 and a Z score of 13.2 and myosin II from Physarum polycephalum (2BL0) with an E value of 3e-38 and a Z score of 11.1(13).

   In conclusion, calmodulin is a calcium-modulated protein that regulates the activity of other proteins. Calmodulin mediates the control of a large number of enzymes, ion channels, and other proteins by using calcium as a sensor and signal transducer. Calmodulin recognizes a variety of target proteins once it binds to calcium and its conformation changes. The EF hands in calmodulin enable it to bind to calcium. Calmodulin belongs to the EFh superfamily, in which the EF hand motif is conserved. There are several other proteins similar to calmodulin in both amino acid sequence and three-dimensional structure. Residues that are critical to calmodulin functions have been identified. Calmodulin is of significant interest to scientist because its ability to bind and regulate a large number of different target proteins, hence influencing many different cellular functions.