Leucine Rich Repeat Kinase 2 (LRRK2) is a large multi-domain kinase which is hyerpactivate in Parkinson’s Disease. When autoinhibited, the catalytic domain of LRRK2 is physically obscured by the N-terminal protein-protein interaction domains, preventing substrate binding. This autoinhibition must be released for kinase activity, but the mechanism by which LRRK2 becomes active remains poorly understood. We propose the use of Designed Ankyrin Repeat Proteins (DARPins) as novel, specific tools to investigate LRRK2 activation and hyperactivation in healthy and disease states. We have characterized the effects of two DARPins, E11 and C12, on the conformation and function of LRRK2 using combined structural and cell biology techniques. Using cryogenic Electron Microscopy in combination with biochemical assays in cells, we determined that DARPins can shift the conformation of LRRK2’s N terminal repeat domains, which leads to altered substrate phosphorylation patterns. For instance, DARPin C12 prevents draping of the N terminal repeat domains over the catalytic domain, resulting in an increase in substrate phosphorylation. Using DARPins as tools, we have therefore characterized a mechanism by which LRRK2’s kinase activity is regulated which has many implications for understanding how PD mutants cause LRRK2 hyperactivation, and for designing novel therapies to treat LRRK2 associated disease.

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