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Biochemical and kinetic properties of the complex Roco G-protein cycle

  • Lina Wauters , Susanne Terheyden , Bernd K. Gilsbach , Margaux Leemans , Panagiotis S. Athanasopoulos , Giambattista Guaitoli , Alfred Wittinghofer , Christian Johannes Gloeckner ORCID logo , Wim Versées EMAIL logo and Arjan Kortholt EMAIL logo
Published/Copyright: September 4, 2018

Abstract

Roco proteins have come into focus after mutations in the gene coding for the human Roco protein Leucine-rich repeat kinase 2 (LRRK2) were discovered to be one of the most common genetic causes of late onset Parkinson’s disease. Roco proteins are characterized by a Roc domain responsible for GTP binding and hydrolysis, followed by a COR dimerization device. The regulation and function of this RocCOR domain tandem is still not completely understood. To fully biochemically characterize Roco proteins, we performed a systematic survey of the kinetic properties of several Roco protein family members, including LRRK2. Together, our results show that Roco proteins have a unique G-protein cycle. Our results confirm that Roco proteins have a low nucleotide affinity in the micromolar range and thus do not strictly depend on G-nucleotide exchange factors. Measurement of multiple and single turnover reactions shows that neither Pi nor GDP release are rate-limiting, while this is the case for the GAP-mediated GTPase reaction of some small G-proteins like Ras and for most other high affinity Ras-like proteins, respectively. The KM values of the reactions are in the range of the physiological GTP concentration, suggesting that LRRK2 functioning might be regulated by the cellular GTP level.

Acknowledgments

We would like to thank H. Pots for the technical assistance. This research was supported by a VUB/UG collaboration agreement (OZR2544; L.W.), the Fonds voor Wetenschappelijk Onderzoek (M.L., W.V.), a Strategic Research Program Financing of the VUB (W.V.), The Michael J. Fox Foundation for Parkinson’s Research (A.K., W.V., C.J.G.), iMed (C.J.G) and a NWO-VIDI grant (A.K.).

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Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2018-0227).


Received: 2018-04-18
Accepted: 2018-07-23
Published Online: 2018-09-04
Published in Print: 2018-11-27

©2018 Walter de Gruyter GmbH, Berlin/Boston

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