Biochemical, kinetic and structural characterization of the unusual GTPase cycle of Roco proteins

Since mutations in the gene coding for the human Roco protein LRRK2 were found to be the most frequent cause of familial Parkinson’s Disease, the protein has been intensively studied. Despite tremendous research efforts, and the discovery of several LRRK2 pathways and interaction partners, still many research questions remain unanswered. One important aspect of the working mechanism of LRRK2 still under debate, is the functioning of the central RocCOR module, responsible for the protein’s GTPase activity.

During my PhD research, I have unravelled the GTP hydrolysis mechanism of Roco proteins. In a first part of the study, I investigated the oligomeric state of the bacterial Chlorobium tepidum Roco protein upon nucleotide binding. We could show that the protein cycles between a monomeric and dimeric state upon nucleotide binding and hydrolysis and that monomerization is a characteristic feature of the GTP hydrolysis cycle. In a second part of the study, I have characterized the GTP hydrolysis mechanism of several prokaryotic Roco proteins and human LRRK2 via biochemical and kinetic experiments. I have shown that the hydrolysis itself is the rate-limiting step in the GTP hydrolysis cycle and that Roco proteins do not require auxiliary proteins for the exchange of nucleotides, although additional factors might still regulate the moderate GTPase activity.

Based on these data, we have proposed a new working hypothesis for Roco proteins in general and LRRK2 in particular. In this way, this work provides valuable new insights in our endeavour to unravel the working mechanism of this protein family.