PROSITE documentation PDOC00018
EF-hand calcium-binding domain signature and profile


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 (DGK) (Ca=2).
  • FAD-dependent glycerol-3-phosphate dehydrogenase (EC 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 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.

Expert(s) to contact by email:

Cox J.A.
Kretsinger R.H.

Last update:

April 2006 / Pattern revised.


Technical section

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

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

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


1AuthorsKawasaki H. Kretsinger R.H.
TitleCalcium-binding proteins 1: EF-hands.
SourceProtein Prof. 2:305-490(1995).
PubMed ID7553064

2AuthorsKretsinger R.H.
TitleCalcium coordination and the calmodulin fold: divergent versus convergent evolution.
SourceCold Spring Harb. Symp. Quant. Biol. 52:499-510(1987).
PubMed ID3454274

3AuthorsMoncrief N.D. Kretsinger R.H. Goodman M.
TitleEvolution of EF-hand calcium-modulated proteins. I. Relationships based on amino acid sequences.
SourceJ. Mol. Evol. 30:522-562(1990).
PubMed ID2115931

4AuthorsNakayama S. Moncrief N.D. Kretsinger R.H.
TitleEvolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories.
SourceJ. Mol. Evol. 34:416-448(1992).
PubMed ID1602495

5AuthorsHeizmann C.W. Hunziker W.
TitleIntracellular calcium-binding proteins: more sites than insights.
SourceTrends Biochem. Sci. 16:98-103(1991).
PubMed ID2058003

6AuthorsKligman D. Hilt D.C.
TitleThe S100 protein family.
SourceTrends Biochem. Sci. 13:437-443(1988).
PubMed ID3075365

7AuthorsStrynadka N.C.J. James M.N.
TitleCrystal structures of the helix-loop-helix calcium-binding proteins.
SourceAnnu. Rev. Biochem. 58:951-998(1989).
PubMed ID2673026

8AuthorsHaiech J. Sallantin J.
TitleComputer search of calcium binding sites in a gene data bank: use of learning techniques to build an expert system.
SourceBiochimie 67:555-560(1985).
PubMed ID3839696

9AuthorsChauvaux S. Beguin P. Aubert J.-P. Bhat K.M. Gow L.A. Wood T.M. Bairoch A.
TitleCalcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D.
SourceBiochem. J. 265:261-265(1990).
PubMed ID2302168

10AuthorsBairoch A. Cox J.A.
TitleEF-hand motifs in inositol phospholipid-specific phospholipase C.
SourceFEBS Lett. 269:454-456(1990).
PubMed ID2401372

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