Conformational scanning of individual EF-hand motifs of calcium sensor protein centrin-1

Centrin-1, a Ca²⁺ sensor protein of the centrin family is a crucial player for cell division in eukaryotes and plays a key role in the microtubule organising centre. Despite being regarded as a calcium sensor with a matched structure to calmodulin/troponin C, the protein undergoes mild changes in conformation and binds Ca²⁺ with moderate affinity. We present an in-depth analysis of the Ca²⁺ sensing by individual EF-hand motifs of centrin-1 and address unsolved questions of the rationales for moderate affinity and conformational transitions of the protein. Employing the more sensitive approach of Trp scanning of individual EF-hand motif, we have undertaken an exhaustive investigation of Ca²⁺ binding to individual EF-hand motifs, named EF1 to EF4. All four EF-hand motifs of centrin-1 are structural as all of them bind both Ca²⁺ and Mg²⁺. EF1 and EF4 are the most flexible sites as they undergo drastic conformational changes following Ca²⁺ binding, whereas EF3 responds to Ca²⁺ minimally. On the other hand, EF2 moves towards the protein surface upon binding Ca²⁺. The independent filling mode of Ca²⁺ to EF-hand motifs and lack of intermotif communication explain the lack of cooperativity of binding, thus constraining centrin-1 to a moderate affinity binding protein. Thus, centrin-1 is distinct from other calcium sensors such as calmodulin.