Deciphering the structural diversity and regulatory layers of expanded Rab GTPases families in the parasitic protist Entamoeba histolytica

Abstract

Rab GTPases are small GTP-binding proteins that play a central role in the regulation of vesicular traffic in all eukaryotes. Pluricellular eukaryotic organisms exhibit an increasing number of Rab GTPase-encoding genes, which may correlate with organismal complexity. However, the genome of the parasitic protist Entamoeba histolytica encodes more than 90 genes for Rab GTPases and other small GTP-binding proteins. The aim of this computational, and hypothesis-driven study was to assess the structural diversity among Rab GTPases of the parasitic protist E. histolytica , in contrast to those of the free-living slime mold Dictyostelium discoideum (28 annotated proteins with more than 40% identity with human or yeast proteins), and to contrast the data against the 63 Rab GTPases in humans. Overall, the vast majority of the encoding Rab GTPases exhibit conserved sequence and fold, with specific exceptions that have long C-termini lacking predicted structure; no homology hits provide hints regarding their function. However, the most important finding is that the GTP-binding residues show greater diversity, and, specifically, the phosphate-binding motif is distinct among the AlphaFold3 models, suggesting that in vivo GTP-associated regulation may vary substantially. Additionally, structural models and structure-based phylogenetic distribution suggest that the parasitic and free-living Rab GTPases may play similar roles, implying that multiple Rab GTPase genes may be involved in fine-tuning vesicular content and transport, thereby shaping protein-sorting mechanisms in single-celled organisms. The computational data presented in this manuscript suggest that even among the highly conserved Rab GTPases, additional layers of regulation may broaden the roles of these enzymes, a possibility that requires extensive experimental characterization and may render them less than ideal drug targets.