Structural Independence of the Two EF-hand Domains of Caltractin

Caltractin (centrin) is a member of the calmodulin subfamily of EF-hand Ca 2+ -binding proteins that is an essential component of microtubule-organizing centers in many organisms ranging from yeast and algae to humans. The protein contains two homologous EF-hand Ca 2+ -binding domains linked by a flexible tether; each domain is capable of binding two Ca 2+ ions. In an effort to search for domain-specific functional properties of caltractin, the two isolated domains were subcloned and expressed in Escherichia coli . Ca 2+ binding affinities and the Ca 2+ dependence of biophysical properties of the isolated domains were monitored by UV, CD, and NMR spectroscopy. Comparisons to the corresponding results for the intact protein showed that the two domains function independently of each other in these assays. Titration of a peptide fragment from the yeast Kar1p protein to the isolated domains and intact caltractin shows that the two domains interact in a Ca 2+ -dependent manner, with the C-terminal domain binding much more strongly than the N-terminal domain. Measurements of the macroscopic Ca 2+ binding constants show that only the N-terminal domain has sufficient apparent Ca 2+ affinity in vitro (1–10 μ m ) to be classified as a traditional calcium sensor in signal transduction pathways. However, investigation of the microscopic Ca 2+ binding events in the C-terminal domain by NMR spectroscopy revealed that the observed macroscopic binding constant likely results from binding to two sites with very different affinities, one in the micromolar range and the other in the millimolar range. Thus, the C-terminal domain appears to also be capable of sensing Ca 2+ signals but is activated by the binding of a single ion.