The Roc‐COR tandem domain of leucine‐rich repeat kinase 2 forms dimers and exhibits conventional Ras‐like GTPase properties.

The Parkinson's disease (PD)‐causative leucine‐rich repeat kinase 2 (LRRK2) belongs to the Roco family of G‐proteins comprising a Ras‐of‐complex (Roc) domain followed by a C‐terminal of Roc (COR) domain in tandem (called Roc‐COR domain). Two prokaryotic Roc‐COR domains have been characterized as 'G proteins activated by guanine nucleotide‐dependent dimerization' (GADs), which require dimerization for activation of their GTPase activity and bind guanine nucleotides with relatively low affinities. Additionally, LRRK2 Roc domain in isolation binds guanine nucleotides with relatively low affinities. As such, LRRK2 GTPase domain was predicted to be a GAD. Herein, we describe the design and high‐level expression of human LRRK2 Roc‐COR domain (LRRK2 Roc‐COR). Biochemical analyses of LRRK2 Roc‐COR reveal that it forms homodimers, with the C‐terminal portion of COR mediating its dimerization. Furthermore, it co‐purifies and binds Mg2+GTP/GDP at 1 : 1 stoichiometry, and it hydrolyzes GTP with Km and kcat of 22 nM and 4.70 × 10−4 min−1, respectively. Thus, even though LRRK2 Roc‐COR forms GAD‐like homodimers, it exhibits conventional Ras‐like GTPase properties, with high‐affinity binding of Mg2+‐GTP/GDP and low intrinsic catalytic activity. The PD‐causative Y1699C mutation mapped to the COR domain was previously reported to reduce the GTPase activity of full‐length LRRK2. In contrast, this mutation induces no change in the GTPase activity, and only slight perturbations in the secondary structure contents of LRRK2 Roc‐COR. As this mutation does not directly affect the GTPase activity of the isolated Roc‐COR tandem, it is possible that the effects of this mutation on full‐length LRRK2 occur via other functional domains. Open Practices: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ The Roc‐COR GTPase supradomain of the Parkinson's disease‐causative kinase LRRK2 (LRRK2 Roc‐COR) consists of a Roc domain and a COR domain arranged in tandem. We generated the recombinant LRRK2 Roc‐COR (inset), which forms homodimers in solution. The C‐terminal portion of COR domain was found to mediate dimerization. Like conventional Ras‐like GTPases, LRRK2 Roc‐COR exhibits tight binding of Mg2+‐guanine nucleotides (inset) and low catalytic efficiency, suggesting that its proper functioning requires a guanine nucleotide exchange factor (GEF) and a GTPase‐activating protein (GAP). LRRK2 Roc‐COR is a useful tool for future investigation to identify GEF(s), GAP(s), and downstream effectors of LRRK2. [ABSTRACT FROM AUTHOR]