The penta-EF hand (PEF) family; The penta-EF hand (PEF) family contains a group of five EF-hand calcium-binding proteins, including several classical calpain large catalytic subunits (CAPN1, 2, 3, 8, 9, 11, 12, 13, 14), two calpain small subunits (CAPNS1 and CAPNS2), as well as non-calpain PEF proteins, ALG-2 (apoptosis-linked gene 2, also termed programmed cell death protein 6, PDCD6), peflin, sorcin, and grancalcin. Based on the sequence similarity of EF1 hand, ALG-2 and peflin have been classified into group I PEF proteins. Calcium-dependent protease calpain subfamily members, sorcin and grancalcin, are group II PEF proteins. Calpains (EC 3.4.22.17) are calcium-activated intracellular cysteine proteases that play important roles in the degradation or functional modulation in a variety of substrates. They have been implicated in a number of physiological processes such as cell cycle progression, remodeling of cytoskeletal-cell membrane attachments, signal transduction, gene expression and apoptosis. ALG-2 is a pro-apoptotic factor that forms a homodimer in the cell or a heterodimer with its closest paralog peflin through their EF5s. Peflin is a 30-kD PEF protein with a longer N-terminal hydrophobic domain than any other member of the PEF family, and it contains nine nonapeptide (A/PPGGPYGGP) repeats. It exists only as a heterodimer with ALG-2. The dissociation of heterodimer occurs in the presence of Ca2+. ALG-2 interacts with various proteins in a Ca2+-dependent manner. Sorcin (for soluble resistance-related calcium binding protein) is a soluble resistance-related calcium-binding protein that participates in the regulation of calcium homeostasis in cells. Grancalcin is a cytosolic Ca2+-binding protein specifically expressed in neutrophils and monocytes/macrophages. It plays a key role in leukocyte-specific functions that are responsible for host defense. Grancalcin can form a heterodimer together with sorcin. Members in this family contain five EF-hand motifs attached to an N-terminal region of variable length containing one or more short Gly/Pro-rich sequences. These proteins form homodimers or heterodimers through pairing between the 5th EF-hands from the two molecules. Unlike calmodulin, the PEF domains do not undergo major conformational changes upon binding Ca2+.

                        10        20        30        40        50        60        70        80
                ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|
gi 15229581  57 NIRSVFESYDNDTNGTIDIEELKKCLEELKLSLSDEEVKGLYSWCDVDGSKGIQFNEFIVLlcLIYLLAkpssesstesr 136
Cdd:cd16185   1 ELRQWFRAVDRDRSGSIDVNELQKALAGGGLLFSLATAEKLIRMFDRDGNGTIDFEEFAAL--HQFLSN----------- 67

                        90       100       110       120       130       140       150
                ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....
gi 15229581 137 emgpklvesifdpIVEVFLFLDKDGKGKLNKADVIKTLNNEDYPLersPSHVTNMRFEEMDWGRKGKVGFREFL 210
Cdd:cd16185  68 -------------MQNGFEQRDTSRSGRLDANEVHEALAASGFQL---DPPAFQALFRKFDPDRGGSLGFDDYI 125

EF-hand, calcium binding motif, found in homologs of mammalian apoptosis-linked gene 2 protein (ALG-2); The family includes some homologs of mammalian apoptosis-linked gene 2 protein (ALG-2) mainly found in lower eukaryotes, such as a parasitic protist Leishmarua major and a cellular slime mold Dictyostelium discoideum. These homologs contains five EF-hand motifs. Due to the presence of unfavorable residues at the Ca2+-coordinating positions, their non-canonical EF4 and EF5 hands may not bind Ca2+. Two Dictyostelium PEF proteins are the prototypes of this family. They may bind to cytoskeletal proteins and/or signal-transducing proteins localized to detergent-resistant membranes named lipid rafts, and occur as monomers or weak homo- or heterodimers like ALG-2. They can serve as a mediator for Ca2+ signaling-related Dictyostehum programmed cell death (PCD).

                        10        20        30        40        50        60        70        80
                ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|
gi 15229581  57 NIRSVFESYDNDTNGTIDIEELKKCLEELKLSLSDEEVKGLYSWCDVDGSKGIQFNEFIVLlcLIYLLAkpssesstesr 136
Cdd:cd16185   1 ELRQWFRAVDRDRSGSIDVNELQKALAGGGLLFSLATAEKLIRMFDRDGNGTIDFEEFAAL--HQFLSN----------- 67

                        90       100       110       120       130       140       150
                ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....
gi 15229581 137 emgpklvesifdpIVEVFLFLDKDGKGKLNKADVIKTLNNEDYPLersPSHVTNMRFEEMDWGRKGKVGFREFL 210
Cdd:cd16185  68 -------------MQNGFEQRDTSRSGRLDANEVHEALAASGFQL---DPPAFQALFRKFDPDRGGSLGFDDYI 125

EF-hand domain pair;

                          10        20        30        40        50        60
                  ....*....|....*....|....*....|....*....|....*....|....*....|
gi 15229581    58 IRSVFESYDNDTNGTIDIEELKKCLE--ELKLSLSDEEVKGLYSWCDVDGSKGIQFNEFI 115
Cdd:pfam13499   4 LKEAFKLLDSDGDGYLDVEELKKLLRklEEGEPLSDEEVEELFKEFDLDKDGRISFEEFL 63

Eps15 homology domain; Pair of EF hand motifs that recognise proteins containing Asn-Pro-Phe (NPF) sequences.

                           10        20        30        40        50        60
                   ....*....|....*....|....*....|....*....|....*....|....*....|...
gi 15229581     61 VFESYDNDTNGTIDIEELKKCLeeLKLSLSDEEVKGLYSWCDVDGSKGIQFNEFIVLLCLIYL 123
Cdd:smart00027  15 IFRSLDKNQDGTVTGAQAKPIL--LKSGLPQTLLAKIWNLADIDNDGELDKDEFALAMHLIYR 75

EF-hand motif found in eukaryotic phosphoinositide-specific phospholipase C (PI-PLC, EC 3.1.4.11) isozymes; PI-PLC isozymes are signaling enzymes that hydrolyze the membrane phospholipids phosphatidylinositol-4,5-bisphosphate (PIP2) to generate two important second messengers in eukaryotic signal transduction cascades, Inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 triggers inflow of calcium from intracellular stores, while DAG, together with calcium, activates protein kinase C, which goes on to phosphorylate other molecules, leading to altered cellular activity. Calcium is required for the catalysis. This family corresponds to the four EF-hand motifs containing PI-PLC isozymes, including PI-PLC-beta (1-4), -gamma (1-2), -delta (1,3,4), -epsilon (1), -zeta (1), eta (1-2). Lower eukaryotes such as yeast and slime molds contain only delta-type isozymes. In contrast, other types of isoforms present in higher eukaryotes. This family also includes 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase 1 (PLC1) from fungi. Some homologs from plants contain only two atypical EF-hand motifs and they are not included. All PI-PLC isozymes except sperm-specific PI-PLC-zeta share a core set of domains, including an N-terminal pleckstrin homology (PH) domain, four atypical EF-hand motifs, a PLC catalytic core, and a single C2 domain. PI-PLC-zeta lacks the PH domain. The PLC catalytic core domain is a TIM barrel with two highly conserved regions (X and Y) split by a highly degenerate linker sequence. Most of EF-hand motifs found in PI-PLCs consist of a helix-loop-helix structure, but lack residues critical to metal binding. Moreover, the EF-hand region of most of PI-PLCs may have an important regulatory function, but it has yet to be identified. However, PI-PLC-zeta is a key exception. It is responsible for Ca2+ oscillations in fertilized oocytes and exhibits a high sensitivity to Ca2+ mediated through its EF-hand domain. In addition, PI-PLC-eta2 shows a canonical EF-loop directing Ca2+-sensitivity and thus can amplify transient Ca2+ signals. Also it appears that PI-PLC-delta1 can regulate the binding of PH domain to PIP2 in a Ca2+-dependent manner through its functionally important EF-hand domains. PI-PLCs can be activated by a variety of extracellular ligands, such as growth factors, hormones, cytokines and lipids. Their activation has been implicated in tumorigenesis and/or metastasis linked to migration, proliferation, growth, inflammation, angiogenesis and actin cytoskeleton reorganization. PI-PLC-beta isozymes are activated by G-protein coupled receptor (GPCR) through different mechanisms. However, PI-PLC-gamma isozymes are activated by receptor tyrosine kinase (RTK), such as Rho and Ras GTPases. In contrast, PI-PLC-epsilon are activated by both GPCR and RTK. PI-PLC-delta1 and PLC-eta 1 are activated by GPCR-mediated calcium mobilization. The activation mechanism for PI-PLC-zeta remains unclear.

                        10        20        30        40        50        60
                ....*....|....*....|....*....|....*....|....*....|....*....|....
gi 15229581  58 IRSVFESYDNDTNGTIDIEELKKCLEELKLSLSDEEVKGLYSWCDVDGSKGIQFNEFIVLLCLI 121
Cdd:cd15898   2 LRRQWIKADKDGDGKLSLKEIKKLLKRLNIRVSEKELKKLFKEVDTNGDGTLTFDEFEELYKSL 65

centrin; Provisional

                         10        20        30        40        50
                 ....*....|....*....|....*....|....*....|....*....|....*...
gi 15229581   58 IRSVFESYDNDTNGTIDIEELKKCLEELKLSLSDEEVKGLYSWCDVDGSKGIQFNEFI 115
Cdd:PTZ00183  19 IREAFDLFDTDGSGTIDPKELKVAMRSLGFEPKKEEIKQMIADVDKDGSGKIDFEEFL 76

calmodulin; Provisional

                         10        20        30        40        50        60
                 ....*....|....*....|....*....|....*....|....*....|....*....|..
gi 15229581   58 IRSVFESYDNDTNGTIDIEELKKCLEELKLSLSDEEVKGLYSWCDVDGSKGIQFNEFIVLLC 119
Cdd:PTZ00184  86 IKEAFKVFDRDGNGFISAAELRHVMTNLGEKLTDEEVDEMIREADVDGDGQINYEEFVKMMM 147

calmodulin; Provisional

                         10        20        30        40        50        60        70        80
                 ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|
gi 15229581   53 EGLRNIRSVFESYDNDTNGTIDIEELKKCLEELKLSLSDEEVKGLYSWCDVDGSKGIQFNEFIVLlcliyllakpssess 132
Cdd:PTZ00184   8 EQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLTL--------------- 72

                         90       100       110       120       130       140       150       160
                 ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|
gi 15229581  133 tesreMGPKLVES-IFDPIVEVFLFLDKDGKGKLNKADVIKTLNNEDYPLerSPSHVTNMrFEEMDWGRKGKVGFREFLF 211
Cdd:PTZ00184  73 -----MARKMKDTdSEEEIKEAFKVFDRDGNGFISAAELRHVMTNLGEKL--TDEEVDEM-IREADVDGDGQINYEEFVK 144

                 ....
gi 15229581  212 AFMS 215
Cdd:PTZ00184 145 MMMS 148

EF-hand motif found in phosphoinositide phospholipase C delta (PI-PLC-delta); PI-PLC-delta isozymes represent a class of metazoan PI-PLCs that are some of the most sensitive to calcium among all PLCs. Their activation is modulated by intracellular calcium ion concentration, phospholipids, polyamines, and other proteins, such as RhoAGAP. Like other PI-PLC isozymes, PI-PLC-delta isozymes contain a core set of domains, including an N-terminal pleckstrin homology (PH) domain, four atypical EF-hand motifs, a PLC catalytic core, and a single C-terminal C2 domain. The PLC catalytic core domain is a TIM barrel with two highly conserved regions (X and Y) split by a highly degenerate linker sequence. There are three PI-PLC-delta isozymes (1, 3 and 4). PI-PLC-delta1 is relatively well characterized. It is activated by high calcium levels generated by other PI-PLC family members, and therefore functions as a calcium amplifier within the cell. Different PI-PLC-delta isozymes have different tissue distribution and different subcellular locations. PI-PLC-delta1 is mostly a cytoplasmic protein, PI-PLC-delta3 is located in the membrane, and PI-PLC-delta4 is predominantly detected in the cell nucleus. PI-PLC-delta isozymes is evolutionarily conserved even in non-mammalian species, such as yeast, slime molds and plants.

                        10        20        30        40        50
                ....*....|....*....|....*....|....*....|....*....|....*...
gi 15229581  58 IRSVFESYDNDTNGTIDIEELKKCLEELKLSLSDEEVKGLYSWCDVDGSKGIQFNEFI 115
Cdd:cd16202   2 LKDQFRKADKNGDGKLSFKECKKLLKKLNVKVDKDYAKKLFQEADTSGEDVLDEEEFV 59

Penta-EF hand, calcium binding motifs, found in calpain-12 (CAPN12); CAPN12, also termed calcium-activated neutral proteinase 12 (CANP 12), is a calpain large subunit mainly expressed in the cortex of the hair follicle. It may affect apoptosis regulation. CAPN12 contains a short N-terminal anchor helix, followed by a calpain cysteine protease (CysPc) domain, a C2-domain-like (C2L) domain, and a C-terminal Ca2+-binding penta-EF-hand (PEF) domain.

                        10        20        30        40        50        60        70
                ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*.
gi 15229581  48 FPKLREGLRNIRSVFESYDNDTNGTIDIEELKKCLEELKLSLSDEEVKGLYSWCDVDGSKgIQFNEFivLLCLIYL 123
Cdd:cd16194  65 FQQLWGYLLEWQAIFTKFDEDTSGTMDSYELRLALNAAGFHLNNQLTETLTSRYRDSRLR-VDFESF--LSCLAHL 137

EF-hand, calcium binding motif, found in beta-parvalbumin; Beta-parvalbumin, also termed Oncomodulin-1 (OM), is a small calcium-binding protein that is expressed in hepatomas, as well as in the blastocyst and the cytotrophoblasts of the placenta. It is also found to be expressed in the cochlear outer hair cells of the organ of Corti and frequently expressed in neoplasms. Mammalian beta-parvalbumin is secreted by activated macrophages and neutrophils. It may function as a tissue-specific Ca2+-dependent regulatory protein, and may also serve as a specialized cytosolic Ca2+ buffer. Beta-parvalbumin acts as a potent growth-promoting signal between the innate immune system and neurons in vivo. It has high and specific affinity for its receptor on retinal ganglion cells (RGC) and functions as the principal mediator of optic nerve regeneration. It exerts its effects in a cyclic adenosine monophosphate (cAMP)-dependent manner and can further elevate intracellular cAMP levels. Moreover, beta-parvalbumin is associated with efferent function and outer hair cell electromotility, and can identify different hair cell types in the mammalian inner ear. Beta-parvalbumin is characterized by the presence of three consecutive EF-hand motifs (helix-loop-helix) called AB, CD, and EF, but only CD and EF can chelate metal ions, such as Ca2+ and Mg2+. The EF site displays a high-affinity for Ca2+/Mg2+, and the CD site is a low-affinity Ca2+-specific site. In addition, beta-parvalbumin is distinguished from other parvalbumins by its unusually low isoelectric point (pI = 3.1) and sequence eccentricities (e.g., Y57-L58-D59 instead of F57-I58-E59).

                        10        20        30        40        50        60
                ....*....|....*....|....*....|....*....|....*....|....*....|...
gi 15229581  58 IRSVFESYDNDTNGTIDIEELKKCLEELKLS---LSDEEVKGLYSWCDVDGSKGIQFNEFIVL 117
Cdd:cd16255  36 VKKVFEIIDQDKSGFIEEEELKLFLQNFSSGareLTDAETKAFLKAGDSDGDGKIGVEEFQAL 98

EF-hand, calcium binding motif, found in apoptosis-linked gene 2 protein (ALG-2) and similar proteins; ALG-2, also termed programmed cell death protein 6 (PDCD6), or probable calcium-binding protein ALG-2, is one of the prototypic members of the penta EF-hand protein family. It is a widely expressed calcium-binding modulator protein associated with cell proliferation and death, as well as cell survival. ALG-2 acts as a pro-apoptotic factor participating in T cell receptor-, Fas-, and glucocorticoid-induced programmed cell death, and also serves as a useful molecular marker for the prognosis of cancers. Moreover, ALG-2 functions as a calcium ion sensor at endoplasmic reticulum (ER) exit sites, and modulates ER-stress-stimulated cell death and neuronal apoptosis during organ formation. Furthermore, ALG-2 can mediate the pro-apoptotic activity of cisplatin or tumor necrosis factor alpha (TNFalpha) through the down-regulation of nuclear factor-kappaB (NF-kappaB) expression. It also inhibits angiogenesis through PI3K/mTOR/p70S6K pathway by interacting of vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, nuclear ALG-2 may participate in the post-transcriptional regulation of Inositol Trisphosphate Receptor Type 1 (IP3R1) pre-mRNA at least in part by interacting with CHERP (Ca2+ homeostasis endoplasmic reticulum protein) calcium-dependently. ALG-2 contains five serially repeated EF-hand motifs and interacts with various proteins, including ALG-2-interacting protein X (Alix), Fas, annexin XI, death-associated protein kinase 1 (DAPk1), Tumor susceptibility gene 101 (TSG101), Sec31A, phospholipid scramblase 3 (PLSCR3), the P-body component PATL1, and endosomal sorting complex required for transport (ESCRT)-III-related protein IST1, in a calcium-dependent manner. It forms a homodimer in the cell or a heterodimer with its closest paralog peflin. Among the PEF proteins, ALG-2 can bind three Ca2+ ions through its EF1, EF3, and EF5 hands, where it is unique in that its EF5 hand binds Ca2+ ion in a canonical coordination.

                        10        20        30        40        50
                ....*....|....*....|....*....|....*....|....*....|....*..
gi 15229581  59 RSVFESYDNDTNGTIDIEELKKCLEELKLSLSDEEVKGLYSWCDVDGSKGIQFNEFI 115
Cdd:cd16183  70 QNCFRSFDRDNSGNIDKNELKQALTSFGYRLSDQFYDILVRKFDRQGRGTIAFDDFI 126

EF-hand motif found in phosphoinositide phospholipase C eta 1 (PI-PLC-eta1); PI-PLC-eta1, also termed 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase eta-1, or phospholipase C-eta-1 (PLC-eta-1), or phospholipase C-like protein 3 (PLC-L3), is a neuron-specific PI-PLC that is most abundant in the brain, particularly in the hippocampus, habenula, olfactory bulb, cerebellum, and throughout the cerebral cortex. It is also expressed in the zona incerta and in the spinal cord. PI-PLC-eta1 may perform a fundamental role in the brain. It may also act in synergy with other PLC subtypes. For instance, it is activated via intracellular Ca2+ mobilization and then plays a role in the amplification of GPCR (G-protein-coupled receptor)-mediated PLC-beta signals. In addition, its activity can be stimulated by ionomycin. PI-PLC-eta1 contains an N-terminal pleckstrin homology (PH) domain, four atypical EF-hand motifs, a PLC catalytic core domain, a C2 domain, and a unique C-terminal tail that terminates with a PDZ-binding motif, a potential interaction site for other signaling proteins. The PLC catalytic core domain is a TIM barrel with two highly conserved regions (X and Y) split by a highly degenerate linker sequence. The C-terminal tail harbors a number of proline-rich motifs which may interact with SH3 (Src homology 3) domain-containing proteins, as well as many serine/threonine residues, suggesting possible regulation of interactions by protein kinases/phosphatases.

                        10        20        30        40        50        60
                ....*....|....*....|....*....|....*....|....*....|....*....|....*..
gi 15229581  58 IRSVFESYDNDTNGTIDIEELKKCLEELKLSLSDEEVKGLYSWCDVDGSKG-IQFNEFIVLLCLIYL 123
Cdd:cd16220   2 VKQTFEEADKNGDGLLNIEEIYQLMHKLNVNLPRRKVRQMFQEADTDENQGtLTFEEFCVFYKMMSL 68

EF-hand domain pair;

                          10        20        30        40        50        60
                  ....*....|....*....|....*....|....*....|....*....|....*....|....*...
gi 15229581   147 FDPIVEVFLFLDKDGKGKLNKADVIKTLNNEDYPLERSPSHVTNMrFEEMDWGRKGKVGFREFLFAFM 214
Cdd:pfam13499   1 EEKLKEAFKLLDSDGDGYLDVEELKKLLRKLEEGEPLSDEEVEEL-FKEFDLDKDGRISFEEFLELYS 67

Penta-EF hand, calcium binding motifs, found in sorcin, grancalcin, and similar proteins; The family corresponds to the second group of penta-EF hand (PEF) proteins that includes sorcin, grancalcin, and similar proteins. Sorcin, also termed 22 kDa Ca2+-binding protein, CP-22, or V19, is a soluble resistance-related calcium-binding protein that is expressed in normal mammalian tissues, such as the liver, lungs and heart. It contains a flexible glycine and proline-rich N-terminal extension and five EF-hand motifs that associate with membranes in a calcium-dependent manner. It may harbor three potential Ca2+ binding sites through its EF1, EF2 and EF3 hands. However, binding of only two Ca2+/monomer suffices to trigger the conformational change that exposes hydrophobic regions and leads to interaction with the respective targets. Sorcin forms homodimers through the association of the unpaired EF5 hand. Among the PEF proteins, sorcin is unique in that it contains potential phosphorylation sites by cAMP-dependent protein kinase (PKA), and it can form a tetramer at slightly acid pH values although remaining a stable dimer at neutral pH. Grancalcin (GCA) is a cytosolic Ca2+-binding protein specifically expressed in neutrophils and monocytes/macrophages. It can strongly interact with sorcin to form a heterodimer and further modulate the function of sorcin. GCA exists as homodimers in solution. It contains five EF-hand motifs attached to an N-terminal region of an approximately 50 residue-long segment rich in glycines and prolines. In contrast with sorcin, GCA binds two Ca2+ ions through its EF1 and EF3 hands.

                        10        20        30        40        50        60        70
                ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*
gi 15229581  46 MKFPKLRE---GLRNIRSVFESYDNDTNGTIDIEELKKCLEELKLSLSDEEVKGLYSWCDVDGSkgIQFNEFIVL 117
Cdd:cd16181  57 MGFNEFKElwaALNQWKTTFMQYDRDRSGTVEPQELQQAIRSFGYNLSPQALNVIVKRYSKNGR--ITFDDFVAC 129

EF-hand domain;

                          10        20
                  ....*....|....*....|....*....
gi 15229581    57 NIRSVFESYDNDTNGTIDIEELKKCLEEL 85
Cdd:pfam13405   1 ELREAFKLFDKDGDGKISLEELRKALRSL 29