Engineering a calcium-dependent conformational change in Calbindin D_9k by secondary elements replacement

EF-hand is a common motif in Ca²⁺-binding proteins, some of which present a conformational change upon Ca²⁺-binding, a relevant property for signal transduction. In the present work, we investigated the behavior of Calbindin D_9k, a modulator protein with a high affinity for Ca²⁺ but structurally insensitive to its presence. Its non-canoncal N-terminal EF-hand was replaced by chimeric motifs, containing increasing structural elements from the sensor troponin C SCIII motif. We demonstrated that the loop and helix II were the necessary elements for a conformational change promoted by calcium in chimeric Calbindin D_9k. Fusion of the isolated chimeric motifs to an activity reporter gene showed the loop as the minimal element to promote a conformational change. The discrepancy between these results is discussed in the light of inter-motif interactions and helix I participation in modulating the Ca²⁺ affinity and restricting motif conformation.