Insilico study to unveil the characteristics of pseudoRab GTPases in pathogenic protozoan parasites: Entamoeba...

Entamoeba histolytica (Eh) is a parasite that utilizes atypical vesicular transport system supported by a huge repertoire of pseudoRab GTPases, to cause amoebic liver abscesses. We have identified 57 pseudoRab GTPases and classified them into 3 classes: 17 Class i enzymes considered GDP-locked enzymes due to low affinity for GTP, 20 Class ii enzymes have attenuated or lost GTPase ability, thus considered GTP-locked; Class iii houses 20 functional enzymes. Evolutionary analysis corroborated the G-motif based classification instead of whole sequence because they house large variations. The evolutionary direction cannot be determined, indicating the possibility of simultaneous emergence of the paralogs of the classical Rab GTPase in Eh. The possible evolutionary mechanisms include gene duplication, domain duplication, and subtle substitutions during recombination events. In-depth Molecular dynamic (MD) simulation provided insight into the dynamic behaviour of the non-canonical Rab members. It suggests that Class i member EhRabX31 may have lost affinity to GTP due to the distorted binding pocket formed by divergent G4 motif (AKxD) and remains GDP-locked. However, class ii member EhRabX10 that shows stable GTP binding, may remain GTP-locked due to divergent G3 motif (DTxDM) abrogating GTP hydrolysis capacity. Furthermore, EhRabX10 showed conformation changes in silico wherein Switch II changed conformations to bind to GTP. This pioneering study has provided insights into a family of viable drug candidates owing to their roles in Eh pathology and their uniqueness that makes them specific to target Eh.

The online version contains supplementary material available at 10.1007/s40203-026-00692-7.