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

Centrin-1, a Ca 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 Ca with moderate affinity. We present an in-depth analysis of the Ca 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 Ca 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 Ca and Mg. EF1 and EF4 are the most flexible sites as they undergo drastic conformational changes following Ca binding, whereas EF3 responds to Ca minimally. On the other hand, EF2 moves towards the protein surface upon binding Ca. The independent filling mode of Ca 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.