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

Luca Bombardi,Carolina Conter,Paola Dominici,Marco Pedretti,Alessandra Astegno

doi:10.3390/biom10081142

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.

Highlights

Centrins are calcium (Ca2+)-binding proteins that are ubiquitously expressed in eukaryotes and commonly associated with the microtubule-organizing center (MTOC), which is the centrosome in mammals, the spindle pole body in yeast, and the basal body in ciliated and flagellated cells
In the present study, using an array of complementary biophysical and biochemical approaches, we investigated the molecular properties of the recombinant proteins TgCEN1 and TgCEN2 with the specific aim to elucidate the role of the Ca2+ ion in determining the structural properties of the proteins and to understand the metal binding, thermodynamic, and structural differences between the two isoforms
TgCEN1 and TgCEN2 are 169- and 170-amino-acid proteins with a predicted molecular mass of 19,343 Da and 19,153 Da, respectively, which is in the range of known centrins

Summary

Introduction

Centrins are calcium (Ca2+)-binding proteins that are ubiquitously expressed in eukaryotes and commonly associated with the microtubule-organizing center (MTOC), which is the centrosome in mammals, the spindle pole body in yeast, and the basal body in ciliated and flagellated cells. Only a small percentage of total centrins (~10%) is restricted to MTOCs, and the remaining percentages are distributed more diffusely in other organelles and cellular compartments [1]. Their different intracellular localizations may be associated with the different functions that have been reported. The selective binding of Ca2+ to the EF-hands of centrin usually causes a conformational change from an apo (closed) state to a holo (open) state causing solvent exposure of a hydrophobic region that is responsible for molecular recognition and interaction with cellular targets. A major difference between CaM and centrins is the presence of an extended unstructured 20–25 residues at the N-terminal domain of centrins that may have specialized a biological function in self-assembly and subcellular localization of the protein

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