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Title: Biochemical characterization of highly purified leucine-rich repeat kinases 1 and 2 demonstrates formation of homodimers
Authors: Civiero, Laura
Vancraenenbroeck, Renée
Belluzzi, Elisa
Beilina, Alexandra
Lobbestael, Evy
Reyniers, Lauran
Gao, Fangye
Micetic, Ivan
De Maeyer, Marc
Bubacco, Luigi
Baekelandt, Veerle
Cookson, Mark R
Greggio, Elisa
Taymans, Jean-Marc # ×
Issue Date: 29-Aug-2012
Publisher: Public Library of Sciene
Series Title: PLoS One vol:7 issue:8 pages:e43472
Article number: e43472
Abstract: Leucine-rich repeat kinase 1 and 2 (LRRK1 and LRRK2) are large multidomain proteins containing kinase, GTPase and multiple protein-protein interaction domains, but only mutations in LRRK2 are linked to familial Parkinson's disease (PD). Independent studies suggest that LRRK2 exists in the cell as a complex compatible with the size of a dimer. However, whether this complex is truly a homodimer or a heterologous complex formed by monomeric LRRK2 with other proteins has not been definitively proven due to the limitations in obtaining highly pure proteins suitable for structural characterization. Here, we used stable expression of LRRK1 and LRRK2 in HEK293T cell lines to produce recombinant LRRK1 and LRRK2 proteins of greater than 90% purity. Both purified LRRKs are folded, with a predominantly alpha-helical secondary structure and are capable of binding GTP with similar affinity. Furthermore, recombinant LRRK2 exhibits robust autophosphorylation activity, phosphorylation of model peptides in vitro and ATP binding. In contrast, LRRK1 does not display significant autophosphorylation activity and fails to phosphorylate LRRK2 model substrates, although it does bind ATP. Using these biochemically validated proteins, we show that LRRK1 and LRRK2 are capable of forming homodimers as shown by single-particle transmission electron microscopy and immunogold labeling. These LRRK dimers display an elongated conformation with a mean particle size of 145 Å and 175 Å respectively, which is disrupted by addition of 6M guanidinium chloride. Immunogold staining revealed double-labeled particles also in the pathological LRRK2 mutant G2019S and artificial mutants disrupting GTPase and kinase activities, suggesting that point mutations do not hinder the dimeric conformation. Overall, our findings indicate for the first time that purified and active LRRK1 and LRRK2 can form dimers in their full-length conformation.
URI: 
ISSN: 1932-6203
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Biochemistry, Molecular and Structural Biology Section
Research Group for Neurobiology and Gene Therapy
× corresponding author
# (joint) last author

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