Paramecium calmodulin mutants defective in ion channel regulation associate with melittin in the absence of calcium but require it for tertiary collapse.

Calmodulin (CaM) is a small acidic protein essential to calcium-mediated signal transduction. Conformational change driven by calcium binding controls its selective activation of myriad target proteins. In most well characterized cases, both homologous domains of CaM interact with a target protein. However, physiologically separable roles for the two domains were demonstrated by mutants of Paramecium tetraurelia [Kung, C. et al. (1992) Cell Calcium 13, 413], some of which have altered calcium affinities [Jaren, O. R. et al. (2000) Biochemistry 39, 6881]. To determine whether these mutants can associate with canonical targets in a calcium-dependent manner, their ability to bind melittin was assessed using analytical gel permeation chromatography, analytical ultracentrifugation, and fluorescence spectroscopy. The Stokes radius of wild-type PCaM and 11 of the mutants decreased dramatically upon binding melittin in the presence of calcium. Fluorescence spectra and sedimentation velocity studies showed that melittin bound to wild-type PCaM and mutants in a calcium-independent manner. However, there were domain-specific perturbations. Mutations in the N-domain of PCaM did not affect the spectrum of melittin (residue W19) under apo or calcium-saturated conditions, whereas most of the mutations in the C-domain did. These data are consistent with a calcium-dependent model of sequential target association whereby melittin (i) binds to the C-domain of PCaM in the absence of calcium, (ii) remains associated with the C-domain upon calcium binding to sites III and IV, and (iii) subsequently binds to the N-domain upon calcium binding to sites I and II of CaM, causing tertiary collapse.