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

J Neurochem. 2018 Nov;147(3):409-428. doi: 10.1111/jnc.14566.

Abstract

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/.

Keywords: GTPase; Parkinson's disease; Roco-family proteins; leucine-rich repeat kinase 2; protein kinase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Dimerization
  • Escherichia coli
  • GTP Phosphohydrolases / genetics*
  • GTP Phosphohydrolases / metabolism*
  • Gene Expression Regulation, Enzymologic / genetics
  • Genes, ras / genetics*
  • Guanine Nucleotides / metabolism
  • Humans
  • Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 / chemistry
  • Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 / genetics*
  • Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 / metabolism*
  • Magnesium / metabolism
  • Mice
  • Mutation / genetics
  • Neuropeptides / biosynthesis
  • Neuropeptides / genetics
  • Protein Multimerization
  • Protein Structure, Secondary / genetics
  • Recombinant Proteins
  • rac1 GTP-Binding Protein / biosynthesis
  • rac1 GTP-Binding Protein / genetics

Substances

  • Guanine Nucleotides
  • Neuropeptides
  • Rac1 protein, mouse
  • Recombinant Proteins
  • LRRK2 protein, human
  • Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
  • GTP Phosphohydrolases
  • rac1 GTP-Binding Protein
  • Magnesium