SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

Abstract

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

Distinct Calcium Binding and Structural Properties of Two Centrin Isoforms from Toxoplasma gondii

“…Indeed, binding partners can have an enormous effect on measurements of Ca 2+ affinities since the two binding events are energetically coupled. Consequently, vast increases in Ca 2+ affinities were often observed when the measurements are made in the presence of a partner protein or even just the interaction motif peptide, as in the case of yeast, plant and green algal centrins [7,14,58]. Thus, a site that appears to have low Ca 2+ affinity when measured for the isolated protein, such as for TgCEN2, may well be fully functional.…”

“…Their different intracellular localizations may be associated with the different functions that have been reported. Indeed, centrins are crucial proteins that are involved in many physiological pathways, such as DNA repair, mRNA export, centrosome duplication, and signal transduction [2][3][4][5][6][7]. Although in the yeast Saccharomyces cerevisiae a single centrin is responsible for such functional diversity, various centrin isoforms have been identified in other organisms, comprising higher plants and mammals [8][9][10][11], and biochemical and structural analyses have indicated distinct molecular properties for diverse centrin isoforms [4,6,[12][13][14][15].…”

The interplay of self-assembly and target binding in centrin 1 from Toxoplasma gondii

“…Circular dichroism (CD) measurements were carried out with a Jasco J-1500 spectropolarimeter equipped with a Peltier type thermostated cell holder. Far-UV spectra (200–250 nm) were an average of five accumulations recorded at 25°C with the same parameters as previously described [ 13 , 24 ]. Protein and peptide concentrations were 10–20 µM in 0.1 cm quartz cuvettes.…”

“…6A). Since the free peptide has a predominantly random coil conformation and generally the EF-hand containing proteins do not increase their helical content after binding to their target peptides [12,13,24,[38][39][40][41][42], the higher ellipticity signal observed for the bimolecular complex could be ascribed to induced helicity in P17-XPC upon binding to C-TgCEN1 (as suggested by the difference spectra where the CD spectrum of C-TgCEN1 alone is subtracted from that of C-TgCEN1-P17-XPC complex).The same changes in CD profiles were obtained for the N-lobe in the presence of Ca 2+ , but not upon peptide addition to the apo-domain (Fig. 6B), therefore supporting the hypothesis that the binding of P17-XPC to the N-lobe of TgCEN1 is Ca 2+ -dependent.…”

“…However, increasing evidence establishes that centrins localize not only to centrosomes but also to other cell compartments and organelles [7]. These different localizations have been shown to be essential to the functions of centrin in various biological processes, such as DNA repair, centrosome duplication, mRNA nuclear export, and signal transduction [8][9][10][11][12][13]. Centrins possess a structural organization similar to the prototype Ca 2+ -binding protein calmodulin (CaM) with the presence of two independent domains, namely the N-and the C-terminal domains which are linked by an α-helix; each domain contains two helix-loop-helix motifs, called the EF-hands, which contain potential Ca 2+ -binding sites.…”