EhRabX3 is an atypical amoebic Rab GTPase with tandem G-domains and exhibit a very low catalytic efficiency.

Crystal structure of EhRabX3 reveals the relative orientation of N- and C-terminal G-domains that are constrained by a short linker. The inter-domain interface is stabilized by both polar and non-polar interactions. Although both the domains exhibited same overall fold, only the N-terminal one showed nucleotide bound to its active site.

The nucleotide-bound N-terminal domain shows significant deviation in its functional motifs from the Ras superfamily members. Due to non-conservative substitutions (V71 and K73) in the switch II region, the complete segment changes its conformation and moved away from the active site. The crystal structure of V71A/K73Q provides a possible explanation for the low catalytic efficiency of EhRabX3.

The crystal structure and further biochemical analysis reveals the existence of an intra-molecular disulfide bond that is critical for maintaining the structural integrity and function of EhRabX3.

Disrupting the intra-molecular disulfide bridge in EhRabX3 renders the protein unstable, which could be used as an important tool for deciphering the biological function of this protein in amoebic trophozoites.