Biophysical analysis of an oligomerization-attenuated variant of the Leishmania donovani dynamin-1-like protein.

Chemotherapy is a cornerstone in the battle against leishmaniasis, a neglected tropical disease caused by Leishmania parasites that affects millions worldwide. An alarming number of reports are describing treatment failure with currently available drugs, thereby explaining the dire need for the discovery of novel compounds, preferably with yet unexplored modes of action. In this respect L. donovani dynamin-1 like protein (LdoDLP1) is of interest as mutations in LdoDLP1 were recently shown to confer resistance to a new antileishmanial compound, suggesting it to be a potential drug target. Through a combination of biochemical, structural, and biophysical methods, we were able to show that wild-type LdoDLP1 has a strong inherent propensity to self-assemble into higher-order oligomers. Guided by structural modeling, a selection of nine point mutations (including resistance markers) were screened for oligomerization behavior to generate self-assembly impaired LdoDLP1 mutants that would occur in solution as dimers and/or tetramers. This led to the identification of a double mutant (G354D/R357S) that exhibits significantly altered and reduced, yet not completely abolished, oligomerization behavior. Further characterization of the LdoDLP1 G354D/R357S double mutant using small-angle X-ray scattering (SAXS) revealed that a fraction of the protein population occurs as a dimer in solution. Additionally, SAXS analysis experimentally confirmed that LdoDLP1, like other dynamin-like proteins, lacks the structurally defined pleckstrin homology (PH) domain of classical dynamins but instead possesses an intrinsically disordered B insert, grouping it among the dynamin-like proteins that play key roles in processes such as mitochondrial fission.