Cdc31, the Saccharomyces cerevisiae centrin, is an EF-hand calcium-binding protein essential for the cell division and mRNA nuclear export. We used biophysical techniques to investigate its calcium, magnesium, and protein target binding properties as well as their conformations in solution. We show here that Cdc31 displays one Ca(2+)/Mg(2+) mixed site in the N-terminal domain and two low-affinity Ca(2+) sites in the C-terminal domain. The affinity of Cdc31 for different natural target peptides (from Kar1, Sfi1, Sac3) that we obtained by isothermal titration calorimetry shows weakly Ca(2+), but also Mg(2+) dependence. The characteristics of target surface binding were shown to be similar; we highlight that the 1-4 hydrophobic amino acid motif, in a stable amphipathic α-helix, is critical for binding. Ca(2+) and Mg(2+) binding increase the α-helix content and stabilize the structure. Analysis of small-angle X-ray scattering experiments revealed that N- and C-terminal domains are not individualized in apo-Cdc31; in contrast, they are separated in the Mg(2+) state, creating a groove in the middle of the molecule that is occupied by the target peptide in the liganded form. Consequently, Mg(2+) seems to have consequences on Cdc31's function and could be important to stimulate interactions in resting cells.

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