Identification and characterization of a calmodulin binding domain in the plasma membrane Ca 2+ -ATPase from Trypanosoma equiperdum

• Published in: Molecular and biochemical parasitology 2018-06, Vol.222, p.51-60
• Main Authors: Ramírez-Iglesias, José Rubén, Pérez-Gordones, María Carolina, Del Castillo, Jesús Rafael, Mijares, Alfredo, Benaim, Gustavo, Mendoza, Marta
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Amino Acid Motifs; Animals; Calcium - metabolism; Calmodulin - chemistry; Calmodulin - metabolism; Cell Membrane - chemistry; Cell Membrane - enzymology; Cell Membrane - genetics; Humans; Molecular Docking Simulation; Protein Binding; Protein Structure, Tertiary; Protozoan Proteins - chemistry; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Rats; Rats, Sprague-Dawley; Trypanosoma - chemistry; Trypanosoma - enzymology; Trypanosoma - genetics; Trypanosomiasis - parasitology
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/j.molbiopara.2018.04.005

The plasma membrane Ca -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 IC s of 200 nM. A stable complex of this peptide and TcCaM was formed in the presence of Ca , 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 Ca /CaM complex. Moreover, when the peptide was incubated with CaM and Ca , a blue shift in the tryptophan fluorescence spectrum (from 350 to 329 nm) was observed. Substitutions at W and F , 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 L and W residues might also participate as anchors to form a 1-4-18-22 motif.