Scherffelia dubia Centrin Exhibits a Specific Mechanism for Ca²⁺-Controlled Target Binding

Abstract

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.

The E144 residue of Scherffelia dubia centrin discriminates between the DNA repair protein XPC and the centrosomal protein Sfi1

“…The ITC experiments presented here and in previous work demonstrated that the affinity of centrin is better for XPC than for other targets such as Sfi1 [22,25,30] and transducin β [Grecu and Assairi to be published elsewhere]. This could suggest that the orientation of the centrin‐binding motif of XPC is the best orientation for effective centrin binding.…”

“…Experiments using microcalorimetry have demonstrated that the C‐terminal domain of centrin is sufficient for target binding. However, for algal centrins, the N‐terminal domain also binds targets, but only in the presence of calcium [21,22]. The structures of the N‐terminal domain of C. reinhardtii [21] and human centrins have been solved by NMR [23].…”

Centrins in unicellular organisms: functional diversity and specialization

“…In general, the C-terminal domain of centrin is able to bind target peptides in a Ca 2+ -independent manner (Li et al 2006;Radu et al 2010;Veeraraghavan et al 2002) and is often important for the subcellular localization of centrin protein (Gavet et al 2003;Li et al 2006;Vonderfecht et al 2011), whereas the N-terminal domain typically binds Ca 2+ and target peptides with lower affinity (Li et al 2006; Thompson et al 2006;Veeraraghavan et al 2002;Yang et al 2006) and plays indispensable roles different from the Cterminal domain (Ivanovska and Rose 2001;Vonderfecht et al 2011). Compared to calmodulins, most centrins possess an extension of variable-length N-terminal to the EF hand motifs (Friedberg 2006;Salisbury 1995).…”

10 Algal cell biology – important tools to understand metal and herbicide toxicity

“…Goedhart and Gadella 2004;, gravity sensing and polarized growth (e.g. Braun and Limbach 2005;Braun and Limbach 2006), exudates secretion (Quesada et al 2006), action potential generation (Beilby 2007) and binding to calcium-binding proteins, for example the basal body connector associated protein centrin (Radu et al 2010). There are very few publications for touch-induced calcium signaling in algae.…”