Project Summary: LRRK2 is a dual enzyme multidomain protein of unknown function. Mutations in LRRK2 has been associated several different diseases including Parkinson’s disease, inflammatory bowel disease, tuberculosis leprosy, and Alzheimer’s disease. The objective of this research program is to determine the molecular mechanisms of action of LRRK2 by using biochemical, biophysical, and chemical biology methods. We have shown that ROC of LRRK2 is a bona fide GTPase, PD-associated mutations trap it in a persistently activated state by impairing GTPase activity, it undergoes nucleotide-dependent conformational changes that is impaired by disease-associated mutations. The proposed studies seek to understand how these conformational changes in the ROC domain regulate the overall structure and activity of LRRK2. The three aims are: Aim 1: To determine the structures of LRRK2 and its conformational dynamics. a) Determine the structural changes in LRRK2 caused by ROC in different nucleotide-bound states and those carrying PD-associated mutations using cryo-EM. Our preliminary single-particle data reveals 2 conformations which may represent the ‘on’ and ‘off’ states. We will define these structures in the proposed studies. b) Define the atomic structures and interactions of LRRK2 by using X-ray crystallography, which is ideal for defining atoms and bonds. Conditions yielding diffracting crystals of full-length LRRK2 have been identified. c) Define the dynamic interactions of residue R1398 in ROC with the γ-phosphate of GTP using NMR, which is key in regulating mechanism of LRRK2 activity. Aim 2: To define the activities of LRRK2. a) To define the regulation of GTPase activity using measuring GTPase activity of constructs carrying PD- mutations, phosphor-memetic, and engineered conformation-constraining disulfide bonds. b) To determine the kinase active and inactive conformation of LRRK2 by measuring kinase activity of conformation-stabilized constructs. c) To characterize the activity and localization of different conformations of LRRK2 in cell models. Aim 3: To define the conformation-activity relationship of LRRK2. a) To modulate LRRK2 conformation using chemical means by employing high throughput chemical screening and chemical biology methods. b) To engineer biologics and potential therapeutic antibodies for trapping the conformations of LRRK2 by using phage-display screening methods.