Scherffelia dubia centrin exhibits a specific mechanism for Ca(2+)-controlled target binding.
Centrins are calcium binding proteins that belong to the EF-hand (or calmodulin) superfamily, which are highly conserved among eukaryotes. Herein, we report the molecular features and binding properties of the green alga Scherffelia dubia centrin (SdCen), a member of the Chlamydomonas reinhardtii centrin (CrCen) subfamily. The Ca(2+) binding capacity of SdCen and its isolated N- and C-terminal domains (N-SdCen and C-SdCen, respectively) was investigated using flow dialysis and isothermal titration calorimetry. In contrast with human centrin 1 and 2 (from the same subfamily), but like CrCen, SdCen exhibits three physiologically significant Ca(2+) binding sites, two in the N-terminal domain and one in the C-terminal domain. Mg(2+) ions could compete with Ca(2+) in one of the N-terminal sites. When Ca(2+) binds, the N-terminal domain becomes more stable and exposes a significant hydrophobic surface that binds hydrophobic fluorescent probes. The Ca(2+) binding properties and the metal ion-induced structural changes in the C-terminal domain are comparable to those of human centrins. We used isothermal titration calorimetry to quantify the binding of SdCen, N-SdCen, and C-SdCen to three types of natural target peptides, derived from the human XPC protein (P17-XPC), the human Sfi1 protein (R17-hSfi1), and the yeast Kar1 protein (P19-Kar1). The three peptides possess the complete (P17-XPC and R17-hSfi1) or partial (P19-Kar1) centrin binding motif (W(1)L(4)L(8)). The integral SdCen exhibits two binding sites for each target peptide, with distinct affinities for each site and each peptide. The high-affinity peptide binding site corresponds to the C-terminal domain of SdCen and displays binding constants and the poor Ca(2+) sensitivities similar to those observed for human centrins. The low-affinity site constituted by the N-terminal domain is active only in the presence of Ca(2+). The thermodynamic binding parameters suggest that the C-terminal domain of SdCen may be constitutively bound to a target, while the N-terminal domain could bind a target only after a Ca(2+) signal. SdCen is also able to interact with calmodulin binding peptides (W(1)F(5)V(8)F(14) motif) with a 1:1 stoichiometry, whereas the isolated N- and C-terminal domains have a much lower affinity. These data suggest particular molecular mechanisms used by SdCen (and probably by other algal centrins) to respond to cellular Ca(2+) signals.
Radu
Laura
L
Institut Curie Centre de Recherche, Centre Universitaire Paris-Sud, 91405 Orsay Cedex, France.
Durussel
Isabelle
I
Assairi
Liliane
L
Blouquit
Yves
Y
Miron
Simona
S
Cox
Jos A
JA
Craescu
Constantin T
CT
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology*; D000460:Chlorophyta / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008555:Melitten / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity; D013816:Thermodynamics; D015662:Trimethoprim, Sulfamethoxazole Drug Combination / Q000737:chemistry* / Q000378:metabolism*
D005656:Fungal Proteins; D008555:Melitten; D015662:Trimethoprim, Sulfamethoxazole Drug Combination; D008274:Magnesium; D002118:Calcium