title: Binding of calcium, magnesium, and target peptides to Cdc31, the centrin of yeast Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: Institut Curie Centre de Recherche, Centre Universitaire Paris-Sud, 91405 Orsay Cedex, France. simona.miron@cea.fr
authors.forename: Simona
authors.initials: S
authors.lastname: Miron
authors.forename: Dominique
authors.initials: D
authors.lastname: Durand
authors.forename: Claudia
authors.initials: C
authors.lastname: Chilom
authors.forename: Javier
authors.initials: J
authors.lastname: Pérez
authors.forename: Constantin T
authors.initials: CT
authors.lastname: Craescu
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013816:Thermodynamics
chemical_list: C083769:CDC31 protein, S cerevisiae; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D008274:Magnesium; D002118:Calcium