Bioinformatic Analysis of Two TOR (Target of Rapamycin)-Like Proteins Encoded by Entamoeba histolytica Revealed Structural Similarities with Functional Homologs

Abstract

The target of rapamycin (TOR), also known as FKBP-rapamycin associated protein (FRAP), is a protein kinase belonging to the PIKK (phosphatidylinositol 3-kinase (PI3K)-related kinases) family. TOR kinases are involved in several signaling pathways that control cell growth and proliferation. Entamoeba histolytica, the protozoan parasite that causes human amoebiasis, contains two genes encoding TOR-like proteins: EhFRAP and EhTOR2. To assess their potential as drug targets to control the cell proliferation of E. histolytica, we studied the structural features of EhFRAP and EhTOR2 using a biocomputational approach. The overall results confirmed that both TOR amoebic homologs share structural similarities with functional TOR kinases, and show inherent abilities to form TORC complexes and participate in protein-protein interaction networks. To our knowledge, this study represents the first in silico characterization of the structure-function relationships of EhFRAP and EhTOR2.

Keywords: Entamoeba histolytica; FKBP-rapamycin associated protein; homology-based protein modeling; structure-function biocomputational analysis; target of rapamycin.

Figure 1

Schematic representation of the canonical domain organization of TOR proteins. HEAT repeats (purple), FAT (dark blue), RBD (light blue), PIKKc (green), and FATC (dark blue). Drawing generated with the IBS program [23].

Figure 2

Schematic representation of the EhFRAP (A) and EhTOR2 (B) domain organization. Protein features were identified by primary structure analysis. While the tetratricopeptide repeats (TPR) are in yellow, the colors for HEAT, FAT and FATC, RBD, and PIKKc were as in Figure 1. Drawings generated with the IBS program.

Figure 3

Multiple sequence alignment of rapamycin-binding domains. Reference sequence (range): mTOR (2015–113), EhFRAP (2002–2105), EhTOR2 (1841–1944), ScTOR1p (1952–2049), and ScTOR2p (1955–2052). Identical residues are within boxes, while those which are similar are shaded (gray). The top-ruler residue-numbering corresponds to the human mTOR sequence.

Figure 4

Multiple sequence alignment of PIKKc domains. Reference sequence (range): mTOR (2183–2430), EhFRAP (2176–2422), EhTOR2 (2019–2260), ScTOR1p (2120–2366), and ScTOR2p (2124–2370). The sequence of functional loops is within colored boxes: catalytic (red) and activation (green). Similar/identical residues, reference sequences, and top-ruler numbers are as described in Figure 3.

Figure 5

Predicted tertiary structure for the rapamycin-binding domain (RBD) of both TOR-like amoebic proteins. Best 3D model (ribbon representation) for the RBD of EhFRAP (A) and EhTOR2 (C). Rainbow-colored from amino (blue) to carboxy (red). An arrow indicates the respective predicted rapamycin binding-site. Best models for the ligand-binding cleft and residues interacting with rapamycin: EhFRAP (B) and EhTOR2 (D). H1 and H4 show the relative locations of the respective α-helix 1 and α-helix 4. Rapamycin (balls and sticks) is colored by element using the default settings.

Figure 6

Predicted tertiary structure for the kinase domain (PIKKc) of both TOR-like amoebic proteins. Best 3D model (ribbon representation) for the PIKKc: EhFRAP (A) and EhTOR2 (B). Rainbow-colored from amino (blue) to carboxy (red).