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Many calcium-binding proteins belong to the same evolutionary family and share a type of calcium-binding domain known as the EF-hand [1,2,3,4,5]. This type of domain consists of a twelve residue loop flanked on both side by a twelve residue α-helical domain (see <PDB:1CLL>). In an EF-hand loop the calcium ion is coordinated in a pentagonal bipyramidal configuration. The six residues involved in the binding are in positions 1, 3, 5, 7, 9 and 12; these residues are denoted by X, Y, Z, -Y, -X and -Z. The invariant Glu or Asp at position 12 provides two oxygens for liganding Ca (bidentate ligand). The basic structural/functional unit of EF-hand proteins is usually a pair of EF-hand motifs that together form a stable four-helix bundle domain. The pairing of EF-hand enables cooperativity in the binding of Ca2+ ions.

We list below the proteins which are known to contain EF-hand regions. For each type of protein we have indicated between parenthesis the total number of EF-hand regions known or supposed to exist. This number does not include regions which clearly have lost their calcium-binding properties, or the atypical low-affinity site (which spans thirteen residues) found in the S-100/ ICaBP family of proteins [6].

• Aequorin and Renilla luciferin binding protein (LBP) (Ca=3).
• α actinin (Ca=2).
• Calbindin (Ca=4).
• Calcineurin B subunit (protein phosphatase 2B regulatory subunit) (Ca=4).
• Calcium-binding protein from Streptomyces erythraeus (Ca=3?).
• Calcium-binding protein from Schistosoma mansoni (Ca=2?).
• Calcium-binding proteins TCBP-23 and TCBP-25 from Tetrahymena thermophila (Ca=4?).
• Calcium-dependent protein kinases (CDPK) from plants (Ca=4).
• Calcium vector protein from amphoxius (Ca=2).
• Calcyphosin (thyroid protein p24) (Ca=4?).
• Calmodulin (Ca=4, except in yeast where Ca=3).
• Calpain small and large chains (Ca=2).
• Calretinin (Ca=6).
• Calcyclin (prolactin receptor associated protein) (Ca=2).
• Caltractin (centrin) (Ca=2 or 4).
• Cell Division Control protein 31 (gene CDC31) from yeast (Ca=2?).
• Diacylglycerol kinase (EC 2.7.1.107) (DGK) (Ca=2).
• FAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) from mammals (Ca=1).
• Fimbrin (plastin) (Ca=2).
• Flagellar calcium-binding protein (1f8) from Trypanosoma cruzi (Ca=1 or 2).
• Guanylate cyclase activating protein (GCAP) (Ca=3).
• Inositol phospholipid-specific phospholipase C isozymes γ-1 and delta-1 (Ca=2) [10].
• Intestinal calcium-binding protein (ICaBPs) (Ca=2).
• MIF related proteins 8 (MRP-8 or CFAG) and 14 (MRP-14) (Ca=2).
• Myosin regulatory light chains (Ca=1).
• Oncomodulin (Ca=2).
• Osteonectin (basement membrane protein BM-40) (SPARC) and proteins that contains an 'osteonectin' domain (QR1, matrix glycoprotein SC1) (see the entry <PDOC00535>) (Ca=1).
• Parvalbumins α and β (Ca=2).
• Placental calcium-binding protein (18a2) (nerve growth factor induced protein 42a) (p9k) (Ca=2).
• Recoverins (visinin, hippocalcin, neurocalcin, S-modulin) (Ca=2 to 3).
• Reticulocalbin (Ca=4).
• S-100 protein, α and β chains (Ca=2).
• Sarcoplasmic calcium-binding protein (SCPs) (Ca=2 to 3).
• Sea urchin proteins Spec 1 (Ca=4), Spec 2 (Ca=4?), Lps-1 (Ca=8).
• Serine/threonine specific protein phosphatase rdgc (EC 3.1.3.16) from Drosophila (Ca=2).
• Sorcin V19 from hamster (Ca=2).
• Spectrin α chain (Ca=2).
• Squidulin (optic lobe calcium-binding protein) from squid (Ca=4).
• Troponins C; from skeletal muscle (Ca=4), from cardiac muscle (Ca=3), from arthropods and molluscs (Ca=2).

There has been a number of attempts [7,8] to develop patterns that pick-up EF-hand regions, but these studies were made a few years ago when not so many different families of calcium-binding proteins were known. We therefore developed a new pattern which takes into account all published sequences. This pattern includes the complete EF-hand loop as well as the first residue which follows the loop and which seem to always be hydrophobic. We also developed a profile that covers the loop and the two α helices.

Positions 1 (X), 3 (Y) and 12 (-Z) are the most conserved.

The 6th residue in an EF-hand loop is, in most cases a Gly, but the number of exceptions to this 'rule' has gradually increased and we felt that the pattern should include all the different residues which have been shown to exist in this position in functional Ca-binding sites.

The pattern will, in some cases, miss one of the EF-hand regions in some proteins with multiple EF-hand domains.

Cox J.A.
Kretsinger R.H.

April 2006 / Pattern revised.

PROSITE methods (with tools and information) covered by this documentation:

EF_HAND_2, PS50222; EF-hand calcium-binding domain profile  (MATRIX)

Sequences known to belong to this class detected by the profile: ALL Other sequence(s) detected in Swiss-Prot: NONE.

• Domain architecture view of Swiss-Prot proteins matching PS50222 • Retrieve an alignment of Swiss-Prot true positive hits:   Clustal format, color, condensed view  / Clustal format, color  / Clustal format, plain text  / Fasta format • Retrieve the sequence logo from the alignment • Taxonomic tree view of all Swiss-Prot/TrEMBL entries matching PS50222 • Retrieve a list of all Swiss-Prot/TrEMBL entries matching PS50222 • Scan Swiss-Prot/TrEMBL entries against PS50222 • view ligand binding statistics

Matching PDB structures: 1A03 1A29 1A2X 1A75 ... [ALL]

EF_HAND_1, PS00018; EF-hand calcium-binding domain  (PATTERN)

Consensus pattern: D-{W}-[DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]-{GP}-[LIVMC]-[DENQSTAGC]-x(2)-[DE]-[LIVMFYW] Sequences known to belong to this class detected by the profile: ALL. for a few sequences Other sequence(s) detected in Swiss-Prot: NONE. probably not calcium-binding and a few proteins for which we have reason to believe that they bind calcium: a number of endoglucanases and a xylanase from the cellulosome complex of Clostridium [9].

• Retrieve an alignment of Swiss-Prot true positive hits:   Clustal format, color, condensed view  / Clustal format, color  / Clustal format, plain text  / Fasta format • Retrieve the sequence logo from the alignment • Taxonomic tree view of all Swiss-Prot/TrEMBL entries matching PS00018 • Retrieve a list of all Swiss-Prot/TrEMBL entries matching PS00018 • Scan Swiss-Prot/TrEMBL entries against PS00018 • view ligand binding statistics

Matching PDB structures: 1A03 1A29 1A2X 1A75 ... [ALL]

1	Authors	Kawasaki H., Kretsinger R.H.
	Title	Calcium-binding proteins 1: EF-hands.
	Source	Protein Prof. 2:305-490(1995).
	PubMed ID	7553064

2	Authors	Kretsinger R.H.
	Title	Calcium coordination and the calmodulin fold: divergent versus convergent evolution.
	Source	Cold Spring Harb. Symp. Quant. Biol. 52:499-510(1987).
	PubMed ID	3454274

3	Authors	Moncrief N.D., Kretsinger R.H., Goodman M.
	Title	Evolution of EF-hand calcium-modulated proteins. I. Relationships based on amino acid sequences.
	Source	J. Mol. Evol. 30:522-562(1990).
	PubMed ID	2115931

4	Authors	Nakayama S., Moncrief N.D., Kretsinger R.H.
	Title	Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories.
	Source	J. Mol. Evol. 34:416-448(1992).
	PubMed ID	1602495

5	Authors	Heizmann C.W., Hunziker W.
	Title	Intracellular calcium-binding proteins: more sites than insights.
	Source	Trends Biochem. Sci. 16:98-103(1991).
	PubMed ID	2058003

6	Authors	Kligman D., Hilt D.C.
	Title	The S100 protein family.
	Source	Trends Biochem. Sci. 13:437-443(1988).
	PubMed ID	3075365

7	Authors	Strynadka N.C.J., James M.N.
	Title	Crystal structures of the helix-loop-helix calcium-binding proteins.
	Source	Annu. Rev. Biochem. 58:951-998(1989).
	PubMed ID	2673026
	DOI	10.1146/annurev.bi.58.070189.004511

8	Authors	Haiech J., Sallantin J.
	Title	Computer search of calcium binding sites in a gene data bank: use of learning techniques to build an expert system.
	Source	Biochimie 67:555-560(1985).
	PubMed ID	3839696

9	Authors	Chauvaux S., Beguin P., Aubert J.-P., Bhat K.M., Gow L.A., Wood T.M., Bairoch A.
	Title	Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D.
	Source	Biochem. J. 265:261-265(1990).
	PubMed ID	2302168

10	Authors	Bairoch A., Cox J.A.
	Title	EF-hand motifs in inositol phospholipid-specific phospholipase C.
	Source	FEBS Lett. 269:454-456(1990).
	PubMed ID	2401372

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