Parasite Genetics and Adaption - Project B5

Structural elucidation of the interaction of apicoplast-resident ferredoxin with its interacting proteins

Host lab: Frank Seeber (FG16, Robert Koch-Institut, Berlin, Germany)

 

Partner lab: Christina Spry & Kevin Saliba (ANU, Research School of Biology, Australia) and Melanie Rug (ANU, Centre for Advance Microscopy, Australia)

 

Background: Ferredoxin (Fd) and its reductase (FNR) arean essential redox system in the apicoplast organelle in both T. gondii and Plasmodium sp. Fd is presumably an important electron donor for the apicoplast-resident synthesis pathways of fatty acids and isoprenoid precursors. However, the precise role of this redox system is ill-defined. In particular, how Fd is capable of selectively passing on electrons via protein-protein interactions to the three different enzymes (LipA, GcpE & LytB) involved in these essential pathways is unknown.

 

Previously, methodology for a two-hybrid screen of genetically encoded cyclic peptides was established in E. coli with the aim of identifying molecules that specifically inhibit the binding of Fd to LipA. While this project is ongoing, we want to complement this approach by screening another type of conformational (de)stabilizer, namely camelid single chain antibodies (nanobodies), for antibody molecules promoting (or preventing) interactions. Previously, nanobodies binding to one or both partners of a complex have been crucial for stabilizing transient complex structures, eventually allowing their 3D-structures to be solved. Currently, no 3D-structure of the complex between PfFd and PfFNR exists. Moreover, a 3D-structure of the transient interaction between Fd and LipA (or LytB or GcpE) would be groundbreaking, allowing us to understand, for the first time, how a small protein like Fd can make specific contacts with such diverse interacting proteins. It would also greatly help in the design of novel drugs binding to Fd.

 

Aims and methods:

  • Identify Fd- and LipA (and/or GcpE, LytB)-specific nanobodies by two-hybrid approach and/or yeast-based platform, based on established and newly prepared nanobody libraries (Berlin).- Characterize the biophysical binding properties of candidate nanobodies to Fd or LipA by differential scanning fluorimetry and isothermal titration calorimetry to reveal binding affinities and localize their binding sites on Fd (RKI and ANU).
  • Attempt to crystalize PfFd/PfFNR with/without bound nanobody to solve structure of complex by crystallography and/or 3D cryo-electron microscopy (both ANU partner labs).

 

Keywords: nanobodies, two-hybrid screening, 3D structure determination, ferredoxin, apicoplast metabolism, protein-protein interaction