Calcium and Magnesium Binding to Human Centrin 3 and Interaction with Target Peptides

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Abstract
There are four isoforms of centrin in mammals, with variable sequence, tissue expression, and functional properties. We have recently characterized a number of structural, ion, and target binding properties of human centrin isoform HsCen2. This paper reports a similar characterization of HsCen3, overexpressed in Escherichia coli and purified by phase-reversed chromatography. Equilibrium and dynamic binding studies revealed that HsCen3 has one mixed Ca2+/Mg2+ binding site of high affinity (Kd = 3 and 10 μM for Ca2+ and Mg2+, respectively) and two Ca2+-specific sites of low affinity (Kd = 140 μM). The metal-free protein is fragmented by an unidentified protease into a polypeptide segment of 11 kDa, which was purified by HPLC, and identified by mass spectrometry as the segment of residues 21−112. Similarly, controlled trypsinolysis on Ca2+-bound HsCen3 yielded a mixture of segments of residues 1−124 and 1−125. The Ca2+/Mg2+ site could be assigned to this segment and thus resides in the N-terminal half of HsCen3. Temperature denaturation experiments, circular dichroism, and utilization of fluorescence hydrophobic probes allowed us to propose that the metal-free protein has molten globule characteristics and that the dication-bound forms are compact with a polar surface for the Mg2+ form and a hydrophobic exposed surface for the Ca2+ form. Thus, HsCen3 could be classified as a Ca2+ sensor protein. In addition, it is able to bind strongly to a model target peptide (melittin), as well as to peptides derived from the protein XPC and Kar1p, with a moderate Ca2+ dependence.