Analysis of structure and stability of Leishmania donovani Rab5a and Rab5b

Area of Science:

• Cell Biology
• Structural Biology
• Parasitology

Background:

• Leishmania donovani Rab5 isoforms, LdRab5a and LdRab5b, are crucial for early endocytosis.
• LdRab5a regulates fluid-phase uptake, while LdRab5b modulates receptor-mediated endocytosis.
• Functional divergence is linked to structural variations in their GTPase domains.

Purpose of the Study:

• To elucidate the structural basis for the distinct functions of LdRab5a and LdRab5b.
• To characterize the active conformation of LdRab5a and compare it with its inactive form.
• To analyze the structure of LdRab5b and its potential conformational dynamics.

Main Methods:

• X-ray crystallography to determine the structures of LdRab5a (GppNHp-bound and GDP-bound) and LdRab5b (GDP-bound).
• Circular Dichroism and Differential Scanning Calorimetry to assess protein folding and stability.
• Molecular dynamics simulations to explore conformational dynamics.
• Comparative structural analysis with homologous proteins.

Main Results:

• The active, GppNHp-bound LdRab5a adopts a closed conformation in Switch I and II regions.
• Guanine nucleotide and Mg2+ binding enhance LdRab5a stability, with the active form showing reduced flexibility.
• LdRab5b structure suggests an intermediate conformation during nucleotide exchange, with lineage-specific adaptations compared to human and plant Rab5 proteins.

Conclusions:

• Structural differences in GTPase domains dictate the distinct roles of LdRab5a and LdRab5b in Leishmania endocytosis.
• The study provides atomic-level insights into the activation mechanism and conformational flexibility of Leishmania Rab5 isoforms.
• Findings contribute to understanding endocytic trafficking regulation in Leishmania and potential therapeutic targets.