All ETDs from UAB

Advisory Committee Chair

Lawrence J Delucas

Advisory Committee Members

Champion Cs Deivanayagam

Debasish Chattopadhyay

John Kappes

Document Type

Dissertation

Date of Award

2016

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Mutations in the leucine rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson disease (PD) , with pathogenic mutations located within each of the two catalytic cores of the protein, the GTPase and kinase domains. The most prevalent pathogenic mutation, G2019S increases kinase activity up to 5-fold, causing significant changes in the protein’s biochemical behavior. Other mutations such as R1441G and I2020T have also been demonstrated to increase LRRK2 kinase activity, however, the detailed mechanisms remains unclear. A major limitation in the field is the lack of structural information of LRRK2. This dissertation detailed the analysis of biophysical and biochemical profiles of different domains of LRRK2, particularly the two enzymatic domains including the kinase domain and the Ras of complex (ROC) GTPase domains. Our result showed that LRRK2 kinase domain is highly unstable and insoluble regardless of the expression system and purification strategies. Sequence analysis and structural modeling of LRRK2 kinase domain suggests the ATP binding pocket of LRRK2 is higher unstable and fluctuated. We therefore designed a mosaic protein where the highly unstable LRRK2 ATP binding pocket is embedded in LRRK2 homologue ROCO4 backbone. The mosaic ROCOC4-LRRK2 resembles LRRK2 kinase domain in the response to different kinase inhibitors, providing a novel tool in LRRK2 inhibitor developing. The ROC GTPase domain was previously found to be phosphorylated by the kinase domain and a reciprocal regulation mechanism was suggested where the kinase activity id dependent on the GTP binding while the phosphorylation of the GTPase domain might affect GTP binding and activity. In this dissertation we studied show phosphorylation of the ROC domain would enhance the GTP hydrolysis activity without significant change in overall structure. Modeling experiments show that phosphorylation induces limited conformational changes at the critical p-loop structure. Finally, ROC appears to be one of many GTPases phosphorylated in p-loop residues, as revealed by alignment of LRRK2 autophosphorylation sites with GTPases annotated in the phosphoproteome database. These results provide an example of a novel mechanism for kinase-mediated control of a GTPase activity.

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