Identification and characterization of a calmodulin binding domain in the plasma membrane Ca2+-ATPase from Trypanosoma equiperdum

Publication date: June 2018Source: Molecular and Biochemical Parasitology, Volume 222Author(s): José Rubén Ramírez-Iglesias, María Carolina Pérez-Gordones, Jesús Rafael del Castillo, Alfredo Mijares, Gustavo Benaim, Marta MendozaAbstractThe 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 200 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 329 nm) was observed. Substitutions at...

Source: Molecular and Biochemical Parasitology - July 5, 2018 Category: Parasitology Source Type: research