Calmodulin-like protein from Entamoeba histolytica: solution structure and calcium-binding properties of a partially folded protein

Abstract

The mechanism of Ca²⁺-signaling in the protozoan parasite Entamoeba histolytica is yet to be understood as many of the key regulators are still to be identified. E. histolytica encodes a number of multi-EF-hand Ca²⁺-binding proteins (EhCaBPs). Functionally only one of these molecules, EhCaBP1, has been characterized to date. The calmodulin-like protein from E. histolytica (abbreviated as EhCaM or EhCaBP3) is a 17.23 kDa monomeric protein that shows maximum sequence identity with heterologous calmodulins (CaMs). Though CaM activity has been biochemically shown in E. histolytica, there are no reports on the presence of a typical CaM. In an attempt to understand the structural and functional similarity of EhCaM with CaM, we have determined the three-dimensional (3D) solution structure of EhCaM using NMR. The EhCaM has a well-folded N-terminal domain and an unstructured C-terminal counterpart. Further, it sequentially binds only two calcium ions, an unusual mode of Ca²⁺-binding among the known CaBPs, notably both in the N-terminal domain of EhCaM. Further, EhCaM is present in the nucleus in addition to the cytoplasm as detected by immunofluorescence staining, unlike other EhCaBPs that are detected only in the cytoplasm. Therefore, this protein is likely to have a different function. The presence of unusual and a diverse set of CaBPs in E. histolytica suggests a distinct Ca²⁺-signaling process in E. histolytica. The results reported here help in understanding the structure−function relationship of CaBPs including their Ca²⁺-binding properties.