Structure-function Studies of Kinetoplastid Proteins

The class kinetoplastida include parasites responsible for devastating diseases like African sleeping sickness, Chagas’s disease and Leishmaniases, mainly effecting people in the developing world. Current treatments are highly toxic and inefficient, leading to an urgent need of novel anti-parasitic compounds. This thesis focuses on the structural characterisation of potential drug targets against these parasites, namely adenosine kinase from Trypanosoma brucei (TbAK), thymidine kinases from T. brucei (TbTK) and Leishmania major (LmTK) and the leucyl aminopeptidases from T. brucei (TbLAP-A), T. cruzi (TcLAP-A) and L. major (LmLAP-A). Structures of TbAK were solved in two conformations, open (apo) and closed (in complex with adenosine and ADP), both to 2.6 A. Comprised of a big α/β-domain and a small lid domain, the structures confirm the large conformational change of the lid domain upon substrate binding. The structures of C-terminally truncated versions of LmTK and TbTK were determined as ligand-bound complexes with resolutions up to 2.4 A and 2.2 A, respectively. They show high similarity to structures of homologues in the PDB. The structures solved in this thesis give valuable information about ligand binding and aid rational drug design. Leucyl aminopeptidase (LAP-A) was evaluated as a potential drug target in T. brucei parasites. It is not essential for T. brucei parasites grown in vitro, shown by generation and analysis of LAP-A-depleted parasites. Although this does not support LAP-A as a drug target in T. brucei, no conclusions can be drawn about the potential in T. cruzi and L. major. Several structures of the LAP-As were solved, the highest resolution ones to 2.3 A, 2.3 A and 2.5 A for TbLAP-A, TcLAP-A and LmLAP-A, respectively. These enzymes are hexameric and show the typical two-domain architecture of M17 LAPs. Although the physiological function remains elusive, the work in this thesis provides a firm basis for future studies.