Cation and peptide binding properties of CML7, a calmodulin-like protein from Arabidopsis thaliana

Trande, Matteo; Pedretti, Marco; Bonza, Maria Cristina; Matteo, Adele Di; D’Onofrio, Mariapina; Dominici, Paola; Astegno, Alessandra

doi:10.1016/j.jinorgbio.2019.110796

Cited by 19 publications

(13 citation statements)

References 74 publications

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“…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.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

Section: Tgcen1 Has a Specific Ca 2+ -Controlled Target Binding Mechanismmentioning

confidence: 99%

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

Conter

Bombardi

Pedretti

et al. 2021

Biochemical Journal

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Tgcen1 Has a Specific Ca 2+ -Controlled Target Binding Mechanismmentioning

confidence: 99%

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

Conter

Bombardi

Pedretti

et al. 2021

Biochemical Journal

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“…Fluorescence emission measurements using 1-anilino-8-naphthalenesulfonic acid (ANS) were conducted on a Jasco FP8200 spectrofluorometer as previously described [ 28 ]. A fixed concentration of 15 μM ANS was mixed with 1 μM protein solution in 50 mM Tris-HCl, 150 mM KCl pH 7.5, in the presence of 2 mM EGTA or 2 mM CaCl 2 .…”

Section: Methodsmentioning

confidence: 99%

“…These spectral changes suggest that the protein undergoes a large Ca 2+ -induced conformational change, likely adopting a somewhat more stable tertiary structure in the presence of Ca 2+ . It is interesting to note that several other centrins [6,14,40], as well as several related Ca 2+ -binding proteins [28,[41][42][43][44], have strikingly similar HSQC NMR spectra with a greater dispersion of cross peaks for the Ca 2+ -loaded state than for the apo-state and with Ca 2+ binding that provides the protein with important structural stability. A similar behavior was also observed for TgCEN2-C, even if upon addition of Ca 2+ the protein appeared to undergo switching with minor changes in conformation compared to TgCEN1-C.…”

Section: Nmr Spectroscopymentioning

confidence: 99%

“…Two new signals in the spectrum of holo-TgCEN1-C and one new peak in the spectrum of holo-TgCEN2-C were clearly detectable in the downfield shifted 1 H region, at 10.74 and 10.81 ppm for TgCEN1-C, and 10.71 ppm for TgCEN2-C, respectively. The signals in the low field-shifted 1 H region generally arise from the invariant glycine residue at the sixth position (Gly-6) of the EF-hand Ca 2+ -binding loop, as a result of Ca 2+ binding [28,41,[43][44][45][46]. Thus, they usually constitute distinctive signs to recognize Ca 2+ -binding sites and to monitor protein folding.…”

Section: Nmr Spectroscopymentioning

confidence: 99%

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

et al. 2020

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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.

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Evaluating the potential of non‐immunosuppressive cyclosporin analogs for targeting Toxoplasma gondii cyclophilin: Insights from structural studies

Favretto,

Jiménez‐Faraco,

Catucci

et al. 2024

Protein Science

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Toxoplasmosis persists as a prevalent disease, facing challenges from parasite resistance and treatment side effects. Consequently, identifying new drugs by exploring novel protein targets is essential for effective intervention. Cyclosporin A (CsA) possesses antiparasitic activity against Toxoplasma gondii, with cyclophilins identified as possible targets. However, CsA immunosuppressive nature hinders its use as an antitoxoplasmosis agent. Here, we evaluate the potential of three CsA derivatives devoid of immunosuppressive activity, namely, NIM811, Alisporivir, and dihydrocyclosporin A to target a previously characterized cyclophilin from Toxoplasma gondii (TgCyp23). We determined the X‐ray crystal structures of TgCyp23 in complex with the three analogs and elucidated their binding and inhibitory properties. The high resolution of the structures revealed the precise positioning of ligands within the TgCyp23 binding site and the details of protein–ligand interactions. A comparison with the established ternary structure involving calcineurin indicates that substitutions at position 4 in CsA derivatives prevent calcineurin binding. This finding provides a molecular explanation for why CsA analogs can target Toxoplasma cyclophilins without compromising the human immune response.

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Cation and peptide binding properties of CML7, a calmodulin-like protein from Arabidopsis thaliana

Cited by 19 publications

References 74 publications

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

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

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

Evaluating the potential of non‐immunosuppressive cyclosporin analogs for targeting Toxoplasma gondii cyclophilin: Insights from structural studies

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