There are several Parkinson’s disease (PD) related proteins that have been defined. Amongst them, leucine-rich repeat kinase 2 (LRRK2) is highly associated to both sporadic and familial PD. LRRK2 consists of multiple protein-protein interaction domains and a catalytic core comprising a RocCOR GTPase domain and a kinase domain. Mutations mapped to RocCOR and the kinase domains are most prevalently found in LRRK2-linked PD patients. Mutations such as G2019S on the kinase domain and R1441C/G on the RocCOR domain contribute to increasing kinase activity. In contrast, the intact RocCOR GTPase domain is required to maintain the kinase activity.
In my research, I have investigated the roles of the RocCOR domain by biochemical and biophysical characterisation. I have demonstrated that LRRK2 RocCOR belongs to conventional small GTPases, requiring a guanine nucleotide exchange factor (GEF) and a GTPase activating protein (GAP).
We used insect cells to express the wild type and also the PD-associated mutant Y1699C recombinant LRRK2 RocCOR domains. After protein purification, we applied circular dichroism to ensure the purified proteins were folded. We can demonstrate the RocCOR domain is catalytically active and its intrinsic activity is relatively low by using the GTPase activity assay. Moreover, the RocCOR domain is able to bind guanine nucleotide relatively tightly as shown by 32P inorganic phosphate in vivo labelling experiment. As the binding of guanine nucleotide to the RocCOR GTPase domain theoretically requires Mg2+ as a cofactor, we introduced inductively coupled plasma-mass spectrometry (ICP-MS) technique to measure the existence of Mg2+. As expected, the stoichiometry of Mg2+ binding to the RocCOR domain was close to 1:1 ratio, suggesting RocCOR domains bind guanine nucleotides tightly. The evidence we provided reveal LRRK2 RocCOR requires upstream regulators such as a GEF and a GAP. Identification of a potential candidate GEF, ARHGEF7 is ongoing.