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Start of funding 01.01.2011

Protein structure and activation mechanism of the cold shock-regulated Protein Kinase A (PKA) of the tropical parasite Trypanosoma

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Protein Kinase A (PKA) is an important regulator of many processes in human and animal cells and is activated by the intracellular messenger molecule cAMP. The PKA of the parasite Trypanosoma, causative agent of African Sleeping Sickness, differs in that it is not activated by cAMP but by cold shock. Such a cold shock occurs naturally during the transmission of the parasite and is important for the differentiation from the mammalian to the insect stage of the trypanosome. The project funded here is focussed on solving the protein structure of the parasite PKA. This will allow comparisons with the human PKA, to elucidate the functional mechanism of cold shock activation and its importance in the parasite’s life cycle. The collaboration will bring together the expertise from Prof. Susan Taylor’s laboratory (San Diego), working on the biochemistry and structural biology of mammalian PKAs, and that from Prof. Michael Bosharts laboratory (Munich) in the field of trypanosome genetics. The project will yield insights into the evolution of PKA and cAMP signalling and the divergent parasite PKA is a potential drug target for the treatment of neglected tropical diseases.

Final report:
The projects aimed at structural information elucidating the unconventional cold shock activation of a cAMP-independent protein kinase A. Recombinant expression the regulatory (R) subunit had been optimized in Munich and was followed by a 5 week working visit of Dr. Daniela Tonn in San Diego, resulting in improvements in purification, upscaling and crystallization assays. Unfortunately, follow-up in San Diego did not provide diffracting crystals and the Taylor group decided to pause the project. In Munich we were able to purify the recombinant R-C holoenzyme and showed kinase activation by alternative ligands. This sheds new light onto the evolution of the PKA family and enables specific pharmacological activation of PKA of pathogenic trypanosomes. The alternative ligands also seem to be important for cold shock-mediated activation. The ligand binding and the cyclic nucleotide binding (CNB)-like pocket were characterized by site-directed mutagenesis and modelled with the human PKA as template. After significant project progress in absence of primary structural data, we would like to return to the original aim to understand the mechanism of activation by cold shock. For practical reasons and current focus of the Taylor lab, we are resuming the crystallisation in collaboration with a local partner at the MPI for Biochemistry. In consequence, we have returned to BaCaTeC the major part of granted funds. We do not exclude a later continuation of our collaboration with San Diego, since with a crystal structure in hands the real work of comparison of the structural basis of PKA activation in different organisms starts.