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Publication Abstract from PubMed

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 degrees 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'-(beta, gamma-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'-(beta, gamma-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.

Structural and Biophysical Characterization of Rab5a from Leishmania Donovani.,Maheshwari D, Yadav R, Rastogi R, Jain A, Tripathi S, Mukhopadhyay A, Arora A Biophys J. 2018 Oct 2;115(7):1217-1230. doi: 10.1016/j.bpj.2018.08.032. Epub 2018, Aug 30. PMID:30241678^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.