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 Å. 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 Å and 2.2 Å, 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 Å, 2.3 Å and 2.5 Å 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.