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

Abstract

Centrins are conserved calcium (Ca2+)-binding proteins typically associated with centrosomes that have been implicated in several biological processes. In Toxoplasma gondii, a parasite that causes toxoplasmosis, three centrin isoforms have been recognized. We have recently characterized the metal binding and structural features of isoform 1 (TgCEN1), demonstrating that it possesses properties consistent with a role as a Ca2+ sensor and displays a Ca2+-dependent tendency to self-assemble. Herein, we expanded our studies, focusing on the self-association and target binding properties of TgCEN1 by combining biophysical techniques including dynamic light scattering, isothermal titration calorimetry, nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy. We found that the self-assembly process of TgCEN1 depends on different physicochemical factors, including Ca2+ concentration, temperature, and protein concentration, and is mediated by both electrostatic and hydrophobic interactions. The process is completely abolished upon removal of the first 21-residues of the protein and is significantly reduced in the presence of a binding target peptide derived from the human XPC protein (P17-XPC). Titration of P17-XPC to the intact protein and isolated domains showed that TgCEN1 possesses two binding sites with distinct affinities and Ca2+ sensitivity; a high-affinity site in the C-lobe which may be constitutively bound to the peptide and a low-affinity site in the N-lobe which is active only upon Ca2+ stimulus. Overall, our results suggest a specific mechanism of TgCEN1 for Ca2+-modulated target binding and support a N-to-C self-assembly mode, in which the first 21-residues of one molecule likely interact with the C-lobe of the other.

Structural Basis for the Functional Diversity of Centrins: A Focus on Calcium Sensing Properties and Target Recognition

“…Studies on centrins have suggested that Ca 2+ -induced polymerization appears to depend on this amino-terminal domain [60]. In both HsCEN2 and TgCEN1, this N-terminal extension is needed for self-assembly induced by Ca 2+ [60][61][62][63]. [48][49][50]62] Homo sapiens HsCEN3 O15182 3…”

“…Thus, HsCEN2 and CDC31 have the ability to bind targets in their C-terminal via a hydrophobic pocket, even independently of Ca 2+ [19,21,49,52,64,69,76]. Notwithstanding, for some centrins, target binding through the N-terminal domain has also been documented [52,63].…”

Conformational Plasticity of Centrin 1 from Toxoplasma gondii in Binding to the Centrosomal Protein SFI1

“…Structural studies of centrin interactions have revealed that the basis for the target selectivity of centrins lies in their ability to interact with regions that have the propensity to form an α-helix and contain a hydrophobic triad, namely W 1 xxL 4 xxxL 8 (1–4–8 motif), which can be either in a forward (W 1 L 4 L 8 ) or reverse (L 8 L 4 W 1 ) orientation [ 6 , 12 , 18 ]. This sequence has been found in many centrin–target complex structures and validated by various biophysical techniques [ 6 , 12 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ].…”

“…The recombinant TgCEN1 (lacking the first 21 residues) and its N-terminal (N-TgCEN1, 22–94 aa) and C-terminal (C-TgCEN1, 95–169 aa) domains were purified as previously described [ 19 , 31 ]. The TgSFI1 peptides were synthesized by GenScript U.S.A. Inc. (Piscataway, NJ, USA).…”

“…Our previous work has shown that TgCEN1 has biochemical properties consistent with a role as a Ca 2+ sensor, since it is able to expose hydrophobic surfaces for interaction with signaling partners upon Ca 2+ binding [ 19 , 31 ]. Thus, its cellular role should be mediated by binding to target proteins.…”