An EF-hand phage display study of calmodulin subdomain pairing

J Mol Biol. 2000 Feb 18;296(2):473-86. doi: 10.1006/jmbi.1999.3452.


The interaction between the two EF-hands, EF3 and EF4, in the C-terminal domain of vertebrate calmodulin is addressed using an EF-hand phage display library. Significant specificity is observed in the presence of Ca(2+), as EF3-EF4 heterodimers are favored over EF3-EF3 and EF4-EF4 homodimers. Primarily EF4-type (and not EF3-type) amino acids are selected when an EF3 peptide is used as the target and vice versa. The results show that this specificity is promoted by several factors. There are three positions, corresponding to Phe89, Ala102, and Leu105, that are strongly selected as EF3-type hydrophobic residues with an EF4 target. When EF3 is the target peptide, EF4-type residues, Ile125, Tyr138 and Phe141, are selected. Remarkably, this subset consists of the same three residue positions in EF3 or EF4 and seems to be involved in specifying the heterodimer preference in both cases. In addition, electrostatic repulsion between the acidic monomers in an EF4 homodimer may further influence the preferred stability of heterodimers. This hypothesis is based on the observation that positively charged residues are strongly selected at four positions when EF4 is the target. A survey of EF-hand pairs suggests that charge separation is a common way to achieve efficient attraction of Ca(2+) without causing electrostatic repulsion between the subdomains. No significant specificity of binding is observed in the ion free state or in the presence of magnesium as no sequence is preferentially selected. The residues at the interface between the two EF-hands are thus highly optimized for the Ca(2+) bound state. At some residue positions, EF3-type amino acids are chosen with EF3-target in the presence of Ca(2+). These residues are not involved in the preference for heterodimer over homodimer formation, but represent key positions to mutate in the intact domain to stabilize its Ca(2+)-bound state.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacteriophages / genetics
  • Bacteriophages / metabolism
  • Base Sequence
  • Binding Sites
  • Calcium / metabolism
  • Calmodulin / chemistry*
  • Calmodulin / metabolism*
  • Cloning, Molecular
  • Dimerization
  • EF Hand Motifs*
  • Edetic Acid / metabolism
  • Humans
  • Magnesium / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Library*
  • Polymerase Chain Reaction
  • Protein Structure, Tertiary
  • Sequence Analysis, Protein
  • Static Electricity
  • Substrate Specificity


  • Calmodulin
  • Peptide Library
  • Edetic Acid
  • Magnesium
  • Calcium