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

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 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 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.