Structural and Biophysical Characterization of Rab5a from Leishmania Donovani

Abstract

Leishmania donovani possess two isoforms of Rab5 (Rab5a and Rab5b), which are involved in fluid phase and receptor-mediated endocytosis, respectively. We have characterized the solution structure and dynamics of a stabilized truncated LdRab5a mutant. For the purpose of NMR structure determination, protein stability was enhanced by systematically introducing various deletions and mutations. Deletion of hypervariable C-terminal and the 20 residues LdRab5a specific insert slightly enhanced the stability, which was further improved by C107S mutation. The final construct, truncated LdRab5a with C107S mutation, was found to be stable for longer durations at higher concentration, with an increase in melting temperature by 10°C. Solution structure of truncated LdRab5a shows the characteristic GTPase fold having nucleotide and effector binding sites. Orientation of switch I and switch II regions match well with that of guanosine 5'-(β, γ-imido)triphosphate (GppNHp)-bound human Rab5a, indicating that the truncated LdRab5a attains the canonical GTP bound state. However, the backbone dynamics of the P-loop, switch I, and switch II regions were slower than that observed for guanosine 5'-(β, γ-imido)triphosphate (GMPPNP)-bound H-Ras. This dynamic profile may further complement the residue-specific complementarity in determining the specificity of interaction with the effectors. In parallel, biophysical investigations revealed the urea induced unfolding of truncated LdRab5a to be a four-state process that involved two intermediates, I1 and I2. The maximal 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (Bis-ANS) binding was observed for I2 state, which was inferred to have molten globule like characteristics. Overall, the strategy presented would have significant impact for studying other Rab and small GTPase proteins by NMR spectroscopy.

Biophysical and Amyloid Behaviour of a Recombinant Rab1 Isoform of Leishmania Donovani

“…Multiple sequence alignment of rLdRab1 performed using BLAST identi ed the small GTP and P-loop NTPase superfamily domain, the Rab1 family (RabF1-F5), Rab subfamily (RabSF2-SF4), GDI and GEF interaction elements, as de ned for Rab proteins (Fig. S1) [5,10]. S2A).…”

“…Molecular functions of four Rab proteins of L. donovani are experimentally described. [10][11][12]. Biophysical chemistry aspects of only a few protozoan Rabs have been explored [10].…”

G Protein-Coupled Receptors as Potential Intercellular Communication Mediators in Trypanosomatidae

“…Cellular switches like small G-proteins ( Maheshwari et al., 2018 ) or cyclic adenosine monophosphate (cAMP)-dependent cellular transduction systems ( Tagoe et al., 2015 ) are described to play a role in the biology of Leishmania , although the details regarding the precise mechanisms that orchestrate the kinetics of these messengers are poorly understood. Leishmania expresses isoforms of the ACs with a molecular structure that differs to that of superior eukaryotes ( Sánchez et al., 1995 ; Kelly et al., 2021 ).…”

Crystal structure of the GDP-bound GTPase domain of Rab5a fromLeishmania donovani

“…The cloning and expression of stabilized LdRab5a has been described previously (Maheshwari et al, 2018). For stabilization, Q93L, P58D, P59G and C107S mutations and Á60-79 and Á196-235 deletions were incorporated sequentially, with monitoring of the linewidths and dispersion in the 15 N-1 H-HSQC spectrum of the protein over a period of nine or more days.…”

“…However, in the crystal structure only the residues from Ala11 to Leu175 are visible. LdRab5a was recombinantly expressed in Escherichia coli as a GST-fusion protein with a thrombin protease cleavage site between the two proteins, as described previously (Maheshwari et al, 2018). Purification of the protein was peformed as follows.…”