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

Calmodulin (CaM) is an EF‐hand protein composed of two calcium (Ca²⁺)‐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 Ca²⁺‐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, Ca²⁺‐free CaM2/3 is predominantly unfolded, but upon binding Ca²⁺, adopts a monomeric structure composed of two EF‐hand motifs bridged by a short antiparallel β‐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 Ca²⁺‐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 Ca²⁺ binding with a K_d1 = 30 ± 5 μM to EF‐3, and a K_d2 > 1000 μM to EF‐2. Binding of the first equivalent of Ca²⁺ 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 Ca²⁺ affinity exhibited by EF‐2 in the context of CaM2/3.