Biochemical and structural studies on LRRK2 regulation.

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Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. In the last decade several gene loci (PARK 1-16) have been linked to the pathogenesis of PD, among them the PARK8 locus that encodes for the Leucine-rich repeat kinase 2 (LRRK2). LRRK2 has been associated with both, sporadic as well as inherited PD suggesting a central role in disease pathology. LRRK2 has kinase as well as GTPase activity and acts also as a scaffold. Several mutations in LRRK2 have been correlated to familial PD, but despite significant research efforts, little is known about the regulation of this 285 kDa multi-domain phosphoprotein. In this application we want to study the molecular basis of LRRK2 regulation addressing both, enzymatic and scaffolding functions. In particular, the proposed structure function approach will allow us to achieve a better mechanistic understanding of the functional consequences of the PD-relevant mutations. We will aim to solve first high-resolution crystal structures of domains of the human LRRK2 protein. Therefore, we have formed an interdisciplinary team between Kassel University (protein biochemistry, biophysics, cell culture) and the Goethe University Frankfurt (Xray-crytallography, structure determination). Applying innovative concepts like “kinase spines” and employing novel tools, i.e. nanobodies and DARPins, we want to investigate the interplay of the GTPase domain with the protein kinase domain. In addition, we want to study the binding of known interaction partners such as 14-3-3-isoforms and their influence on conformation and activity of LRRK2. We already described an essential function for cAMP-dependent protein kinase (PKA) and 14-3-3 interaction in the negative regulation of LRRK2 kinase activity. We want to investigate the temporal and spatial regulation of the LRRK2/PKA signaling module via A-kinase anchoring proteins (AKAPs). Employing a combination of structural, molecular, biochemical, cellular and biophysical techniques will help us to describe the modulation of LRRK2 function in healthy and diseased states. Understanding LRRK2 regulation on a molecular level will not only provide insights into mechanisms of PD neurodegeneration, but may also facilitate the development of therapeutics.