Evidence of the presence of a calmodulin-sensitive plasma membrane Ca 2+ -ATPase in Trypanosoma equiperdum

• Published in: Molecular and biochemical parasitology 2017-04, Vol.213, p.1-11
• Main Authors: Pérez-Gordones, María Carolina, Ramírez-Iglesias, José Rubén, Cervino, Vincenza, Uzcanga, Graciela L, Benaim, Gustavo, Mendoza, Marta
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Blotting, Western; Calcium-Transporting ATPases - analysis; Calcium-Transporting ATPases - chemistry; Calcium-Transporting ATPases - genetics; Calmodulin - metabolism; Cell Membrane - enzymology; Cloning, Molecular; Immunoassay; Models, Molecular; Prospective Studies; Protein Conformation; Protein Domains; Sequence Alignment; Trypanosoma - enzymology; Trypanosoma - genetics
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/j.molbiopara.2017.02.001

Trypanosoma equiperdum belongs to the subgenus Trypanozoon, which has a significant socio-economic impact by limiting animal protein productivity worldwide. Proteins involved in the intracellular Ca regulation are prospective chemotherapeutic targets since several drugs used in experimental treatment against trypanosomatids exert their action through the disruption of the parasite intracellular Ca homeostasis. Therefore, the plasma membrane Ca -ATPase (PMCA) is considered as a potential drug target. This is the first study revealing the presence of a PMCA in T. equiperdum (TePMCA) showing that it is calmodulin (CaM) sensitive, revealed by ATPase activity, western-blot analysis and immuno-absorption assays. The cloning sequence for TePMCA encodes a 1080 amino acid protein which contains domains conserved in all PMCAs so far studied. Molecular modeling predicted that the protein has 10 transmembrane and three cytoplasmic loops which include the ATP-binding site, the phosphorylation domain and Ca translocation site. Like all PMCAs reported in other trypanosomatids, TePMCA lacks a classic CaM binding domain. Nevertheless, this enzyme presents in the C-terminal tail a region of 28 amino acids (TeC28), which most likely adopts a helical conformation within a 1-18 CaM binding motif. Molecular docking between Trypanosoma cruzi CaM (TcCaM) and TeC28 shows a significant similarity with the CaM-C28PMCA4b reference structure (2kne). TcCaM-TeC28 shows an anti-parallel interaction, the peptide wrapped by CaM and the anchor buried in the hydrophobic pocket, structural characteristic described for similar complexes. Our results allows to conclude that T. equiperdum possess a CaM-sensitive PMCA, which presents a non-canonical CaM binding domain that host a 1-18 motif.