Biochemical and structural characterization of Roco4: A model to understand LRRK2 mediated parkinson´s disease

LRRK2 is a multi-domain protein that belongs to the group of Roco proteins and is activated by dimerization. Since purification of LRRK2 or any fragments of it is a challenging task and all attempts so far only led to insoluble protein or protein that is constantly bound to chaperones we use a different attempt. We make use of Roco4 from D. discoideum which can be purified in amounts suitable for a structural and biochemical characterization. We were able to crystallize the kinase domain and thereby explaining the increase activity of the most prevalent PD mutation. Furthermore we showed that it is possible to co-crystallize LRRK2 specific with the kinase domain to analyze binding on an atomic level.
In pull-down experiments we showed the LRRK2 is dimerizing via its COR domain and that dimerization is essential for the GTPase activity of the protein. Size exclusion experiments with a homolog protein from M. barkeri showed that dimerization is independent of the nucleotide state of the Roc domain and only depends on an intact COR domain.
Further experiments with full-length Roco proteins will reveal the relationship between the kinase and the Roc domain of Roco proteins and explain the complicated regulatory mechanism within Roco proteins.