Calcium-induced folding of a fragment of calmodulin composed of EF-hands 2 and 3

Protein Sci. 2007 Jun;16(6):1119-32. doi: 10.1110/ps.072777107. Epub 2007 May 1.


Calmodulin (CaM) is an EF-hand protein composed of two calcium (Ca(2+))-binding EF-hand motifs in its N-domain (EF-1 and EF-2) and two in its C-domain (EF-3 and EF-4). In this study, we examined the structure, dynamics, and Ca(2+)-binding properties of a fragment of CaM containing only EF-2 and EF-3 and the intervening linker sequence (CaM2/3). Based on NMR spectroscopic analyses, Ca(2+)-free CaM2/3 is predominantly unfolded, but upon binding Ca(2+), adopts a monomeric structure composed of two EF-hand motifs bridged by a short antiparallel beta-sheet. Despite having an "even-odd" pairing of EF-hands, the tertiary structure of CaM2/3 is similar to both the "odd-even" paired N- and C-domains of Ca(2+)-ligated CaM, with the conformationally flexible linker sequence adopting the role of an inter-EF-hand loop. However, unlike either CaM domain, CaM2/3 exhibits stepwise Ca(2+) binding with a K (d1) = 30 +/- 5 microM to EF-3, and a K (d2) > 1000 microM to EF-2. Binding of the first equivalent of Ca(2+) induces the cooperative folding of CaM2/3. In the case of native CaM, stacking interactions between four conserved aromatic residues help to hold the first and fourth helices of each EF-hand domain together, while the loop between EF-hands covalently tethers the second and third helices. In contrast, these aromatic residues lie along the second and third helices of CaM2/3, and thus are positioned adjacent to the loop between its "even-odd" paired EF-hands. This nonnative hydrophobic core packing may contribute to the weak Ca(2+) affinity exhibited by EF-2 in the context of CaM2/3.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Calcium / pharmacology*
  • Calmodulin / chemistry*
  • Calmodulin / genetics
  • Calmodulin / metabolism
  • Carbon Isotopes
  • EF Hand Motifs*
  • Humans
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Sequence Data
  • Nitrogen Isotopes
  • Protein Conformation / drug effects
  • Protein Folding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid


  • Calmodulin
  • Carbon Isotopes
  • Nitrogen Isotopes
  • Calcium

Associated data

  • PDB/2HF5