Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2.

Abstract Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, R as o f c omplex proteins) and kinase domains and a connecting COR ( C -terminus o f R OC) domain. In contrast, the precise functions of the protein–protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR LRRK2 ) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR LRRK2 in Escherichia coli . Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR LRRK2 thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% β-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR LRRK2 multimerization was detected via cross-linking studies. Finally, the LRR LRRK2 clinical mutations did not influence LRR LRRK2 secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR LRRK2 inter- and intramolecular interactions.