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

Abstract The Parkinson's disease ( PD )‐causative leucine‐rich repeat kinase 2 ( LRRK 2) 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’ ( GAD s), which require dimerization for activation of their GTP ase activity and bind guanine nucleotides with relatively low affinities. Additionally, LRRK 2 Roc domain in isolation binds guanine nucleotides with relatively low affinities. As such, LRRK 2 GTP ase domain was predicted to be a GAD . Herein, we describe the design and high‐level expression of human LRRK 2 Roc‐ COR domain ( LRRK 2 Roc‐ COR ). Biochemical analyses of LRRK 2 Roc‐ COR reveal that it forms homodimers, with the C‐terminal portion of COR mediating its dimerization. Furthermore, it co‐purifies and binds Mg 2+ GTP / GDP at 1 : 1 stoichiometry, and it hydrolyzes GTP with K m and k cat of 22 nM and 4.70 × 10 −4 min −1 , respectively. Thus, even though LRRK 2 Roc‐ COR forms GAD ‐like homodimers, it exhibits conventional Ras‐like GTP ase properties, with high‐affinity binding of Mg 2+ ‐ GTP / GDP and low intrinsic catalytic activity. The PD ‐causative Y1699C mutation mapped to the COR domain was previously reported to reduce the GTP ase activity of full‐length LRRK 2. In contrast, this mutation induces no change in the GTP ase activity, and only slight perturbations in the secondary structure contents of LRRK 2 Roc‐ COR . As this mutation does not directly affect the GTP ase activity of the isolated Roc‐ COR tandem, it is possible that the effects of this mutation on full‐length LRRK 2 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/ image