title: Paramecium calmodulin mutants defective in ion channel regulation associate with melittin in the absence of calcium but require it for tertiary collapse.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 52242-1109, USA.
authors.forename: B R
authors.initials: BR
authors.lastname: Sorensen
authors.forename: J T
authors.initials: JT
authors.lastname: Eppel
authors.forename: M A
authors.initials: MA
authors.lastname: Shea
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002147:Calmodulin / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D002850:Chromatography, Gel; D007473:Ion Channels / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D008555:Melitten / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010247:Paramecium / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014461:Ultracentrifugation
chemical_list: D002147:Calmodulin; D007473:Ion Channels; D008555:Melitten; D014364:Tryptophan; D002118:Calcium