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 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 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 mu M to EF-3, and a K-d2 > 1000 mu M 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.