Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

The Roc-COR domain of Leucine-rich repeat kinase 2 belongs to conventional Ras-like GTPases, not a GAD (#203)

Lung-Yu Liang 1 2 3 , Ryan Daryl Mills 1 , Daisy Sio-Seng Lio 1 , George Cao 1 , Yee-Fong Mok 1 4 , Michael Griffin 1 4 , Janetta Culvenor 5 , Vijaya Kenche 6 , Heung-Chin Cheng 1
  1. Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia
  2. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia
  3. Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia
  4. Bio21, University of Melbourne, Melbourne, Victoria, Australia
  5. Department of Pathology, University of Melbourne, Melbourne, Victoria, Australia
  6. The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Victoria, Australia

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder. Lack of understanding of the molecular mechanisms underpinning PD hinders development of early detection and effective therapeutics against PD. Mutations on Leucine-rich repeat kinase 2 (LRRK2) were discovered to be major predisposing factors for both sporadic and inherited PD patients in 2004. LRRK2 is a huge protein encompassing two enzymatic domains and multiple protein-protein interaction domains. In addition to phosphorylating downstream substrates, the LRRK2 kinase domain can autophosphorylate its adjacent Roc-COR GTPase domain, exerting regulatory effects. Reciprocally, binding of guanine nucleotides to the Roc-COR GTPase domain intramolecularly modulates the kinase activity. As the most prevalent PD-associated mutations are mapped to these two catalytic domains, it is important to characterise how they function, and how mutations can dysregulate, and eventually contribute to neurotoxicity. One of the debating issues is whether the LRRK2 Roc-COR GTPase domain is a conventional Ras-like GTPase, or it belongs to G-proteins activated by nucleotide-dependent dimerization (GADs). Biochemical analyses and protein structures of the homologous Roc-COR domains from C. tepidum and M. Barkeri favour the argument that LRRK2 is a GAD. Besides, the isolated Roc domain of LRRK2 exhibiting weak binding affinity of guanine nucleotides also substantiates LRRK2 belongs to GADs. However, as the COR domain C-terminal to the Roc domain is evolutionarily conserved and structurally inseparable, we attempted to characterise the biochemical properties of the intact LRRK2 Roc-COR domain. We successfully generated sufficient amount of recombinant LRRK2 Roc-COR proteins, allowing us to demonstrate that the LRRK2 Roc-COR domain binds guanine nucleotides very tightly (Km ~50 nM), with very low intrinsic GTPase activity (Kcat ~0.013 min-1), similar to the Ras-like GTPases. Substantiated by other evidence, we concluded that LRRK2 is not a GAD, such that it requires upstream regulators to facilitate the GTP hydrolysis and guanine nucleotide exchange.