Identification and characterization of a calmodulin binding domain in the plasma membrane Ca2+-ATPase from Trypanosoma equiperdum.
Mol Biochem Parasitol
; 222: 51-60, 2018 06.
Article
em En
| MEDLINE
| ID: mdl-29752964
The plasma membrane Ca2+-ATPase (PMCA) from trypanosomatids lacks a classical calmodulin (CaM) binding domain, although CaM stimulated activities have been detected by biochemical assays. Recently we proposed that the Trypanosoma equiperdum CaM-sensitive PMCA (TePMCA) contains a potential 1-18 CaM-binding motif at the C-terminal region of the pump. In the present study, we evaluated the potential CaM-binding motifs using CaM from Trypanosoma cruzi and either the recombinant full length TePMCA C-terminal sequence (P14) or synthetic peptides comprising different regions of the C-terminal domain. We demonstrated that P14 and a synthetic peptide corresponding to residues 1037-1062 (which contains the predicted 1-18 binding motif) competed efficiently for binding to TcCaM, exhibiting similar IC50s of 200â¯nM. A stable complex of this peptide and TcCaM was formed in the presence of Ca2+, as determined by native-polyacrylamide gel electrophoresis. A predicted structure obtained by molecular docking showed an interaction of the 1-18 binding motif with the Ca2+/CaM complex. Moreover, when the peptide was incubated with CaM and Ca2+, a blue shift in the tryptophan fluorescence spectrum (from 350 to 329â¯nm) was observed. Substitutions at W1039 and F1056, strongly decreased both CaM-peptide interaction and the complex assembly. Our results demonstrated the presence of a functional 1-18 motif at the TePMCA C-terminal domain. Furthermore, on the basis of spectrofluorometric assays and the resulting structure modeled by docking we propose that the L1042 and W1060 residues might also participate as anchors to form a 1-4-18-22 motif.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Trypanosoma
/
Calmodulina
/
Proteínas de Protozoários
/
Membrana Celular
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Cálcio
/
Adenosina Trifosfatases
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Mol Biochem Parasitol
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Venezuela
País de publicação:
Holanda