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Biochemical Characterization of Highly Purified Leucine-Rich Repeat Kinases 1 and 2 Demonstrates Formation of Homodimers

Authors: CIVIERO, LAURA; Renée Vancraenenbroeck; BELLUZZI, ELISA; Alexandra Beilina; Evy Lobbestael; Lauran Reyniers; Fangye Gao; +7 Authors

Biochemical Characterization of Highly Purified Leucine-Rich Repeat Kinases 1 and 2 Demonstrates Formation of Homodimers

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.

Keywords

ALPHA-SYNUCLEIN, PROTEINS, General Science & Technology, Science, Protein Serine-Threonine Kinases, Leucine-Rich Repeat Proteins, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2, Adenosine Triphosphate, PARKINSONS-DISEASE, Microscopy, Electron, Transmission, DOMINANT PARKINSONISM, Humans, Point Mutation, ROC DOMAIN, Phosphorylation, Chromatography, Science & Technology, Circular Dichroism, Q, Lentivirus, R, Proteins, LRRK2, LEUCINE-RICH-REPEAT-KINASE-2, GENE, Immunohistochemistry, Multidisciplinary Sciences, HEK293 Cells, Microscopy, Fluorescence, Mutation, Science & Technology - Other Topics, Medicine, DISEASE-ASSOCIATED MUTATIONS, Dimerization, GTP-BINDING, Research Article, Protein Binding

  • BIP!
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    citations
    This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
    96
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    This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
    Top 10%
    influence
    This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
    Top 10%
    impulse
    This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
    Top 10%
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citations
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
96
Top 10%
Top 10%
Top 10%
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gold