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

Abstract

Centrins are calcium (Ca2+)-binding proteins that have been implicated in several regulatory functions. In the protozoan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, three isoforms of centrin have been identified. While increasing information is now available that links the function of centrins with defined parasite biological processes, knowledge is still limited on the metal-binding and structural properties of these proteins. Herein, using biophysical and structural approaches, we explored the Ca2+ binding abilities and the subsequent effects of Ca2+ on the structure of a conserved (TgCEN1) and a more divergent (TgCEN2) centrin isoform from T. gondii. Our data showed that TgCEN1 and TgCEN2 possess diverse molecular features, suggesting that they play nonredundant roles in parasite physiology. TgCEN1 binds two Ca2+ ions with high/medium affinity, while TgCEN2 binds one Ca2+ with low affinity. TgCEN1 undergoes significant Ca2+-dependent conformational changes that expose hydrophobic patches, supporting a role as a Ca2+ sensor in toxoplasma. In contrast, Ca2+ binding has a subtle influence on conformational features of TgCEN2 without resulting in hydrophobic exposure, suggesting a different Ca2+ relay mode for this isoform. Furthermore, TgCEN1 displays a Ca2+-dependent ability to self-assemble, while TgCEN2 did not. We discuss our findings in the context of Ca2+ signaling in toxoplasma.

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

“…Indeed, no changes in turbidity at 340 nm were observed upon addition of 1 mM Ca 2+ to 21TgCEN1 sample under the same experimental conditions used for the full-length protein (Fig. S2A) [16]. Accordingly, new DLS measurements showed the presence of a single peak centered at 7.01 ± 0.01 nm compatible with a single prevailing monomeric protein in both the absence and presence of Ca 2+ (Fig.…”

“…The full-length TgCEN1 and the truncated variant of TgCEN1 lacking the first 21 residues (21TgCEN1) were overexpressed in E.coli strain Rosetta and purified as described previously [16]. The N-and C-terminal domains of TgCEN1 were produced by recombinant techniques.…”

“…Our previous studies demonstrated that, upon binding Ca 2+ , TgCEN1 exposed a hydrophobic area which is likely instrumental for the interaction with target proteins [16]. The physiological targets of TgCEN1 are not known at present, although it is well-established that the centrin targets in many organisms usually have a typical binding motif consisting of the hydrophobic triad W 1 L 4 L 8 (1-4-8 motif) [33][34][35].…”

“…Toxoplasma gondii, an apicomplexan parasite responsible for toxoplasmosis, possesses three centrin isoforms, i.e., centrin 1 (TgCEN1), centrin 2 (TgCEN2), and centrin 3 (TgCEN3) [15]. Recent studies in our laboratory demonstrated that TgCEN1 behaves biochemically as a Ca 2+ sensor in vitro, being able to bind two Ca 2+ ions via its N-terminal EF-hands and to undergo significant Ca 2+ -dependent conformational changes that result in the exposure of hydrophobic surfaces [16]. Thus, as a regulatory protein, the biological function of TgCEN1 should be mediated by interaction with downstream proteins.…”

“…Experimental data on centrins from yeast, green algae, ciliate, and humans demonstrated that they can form multimers and suggested that centrins have important structural functions by contributing to the formation of Ca 2+ -sensitive contractile filaments [14,[17][18][19]. Our previous work has shown that TgCEN1 is also able to reversibly selfassemble in the presence of Ca 2+ and has demonstrated the importance of the N-terminal fragment (preceding the first EF-hand motif) in this process [16]. Notably, TgCEN1 mainly localizes to centrioles/centrosomes [15], and therefore it may also be involved in the formation of supramolecular assemblies.…”