title: Conformational heterogeneity of the Roc domains in C. tepidum Roc-COR and implications for human LRRK2 Parkinson mutations.
abstract: Ras of complex proteins (Roc) is a Ras-like GTP-binding domain that always occurs in tandem with the C-terminal of Roc (COR) domain and is found in bacteria, plants and animals. Recently, it has been shown that Roco proteins belong to the family of G-proteins activated by nucleotide (nt)-dependent dimerization (GADs). We investigated the RocCOR tandem from the bacteria Chlorobium tepidum with site-directed spin labelling and pulse EPR distance measurements to follow conformational changes during the Roco G-protein cycle. Our results confirm that the COR domains are a stable dimerization device serving as a scaffold for the Roc domains that, in contrast, are structurally heterogeneous and dynamic entities. Contrary to other GAD proteins, we observed only minor structural alterations upon binding and hydrolysis of GTP, indicating significant mechanistic variations within this protein class. Mutations in the most prominent member of the Roco family of proteins, leucine-rich repeat (LRR) kinase 2 (LRRK2), are the most frequent cause of late-onset Parkinson's disease (PD). Using a stable recombinant LRRK2 Roc-COR-kinase fragment we obtained detailed kinetic data for the G-protein cycle. Our data confirmed that dimerization is essential for efficient GTP hydrolysis and PD mutations in the Roc domain result in decreased GTPase activity. Previous data have shown that these LRRK2 PD-mutations are located in the interface between Roc and COR. Importantly, analogous mutations in the conserved C. tepidum Roc/COR interface significantly influence the structure and nt-induced conformational changes of the Roc domains.
authors: Department of Physics, University of Osnabrueck, Barbarastr. 7, 49076 Osnabrueck, Germany.
authors: Katharina
authors: K
authors: Rudi
authors: Department of Biochemistry, University of Groningen, Nijenborgh 4, Groningen 9747 AG, Netherlands.
authors: Franz Y
authors: FY
authors: Ho
authors: Department of Cell Biochemistry, University of Groningen, Nijenborgh 7, Groningen 9747 AG, Netherlands.
authors: Bernd K
authors: BK
authors: Gilsbach
authors: Department of Cell Biochemistry, University of Groningen, Nijenborgh 7, Groningen 9747 AG, Netherlands.
authors: Henderikus
authors: H
authors: Pots
authors: Structural Biology Group, Max Planck-Institute for Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
authors: Alfred
authors: A
authors: Wittinghofer
authors: Department of Cell Biochemistry, University of Groningen, Nijenborgh 7, Groningen 9747 AG, Netherlands a.kortholt@rug.nl jklare@uos.de.
authors: Arjan
authors: A
authors: Kortholt
authors: Department of Physics, University of Osnabrueck, Barbarastr. 7, 49076 Osnabrueck, Germany a.kortholt@rug.nl jklare@uos.de.
authors: Johann P
authors: JP
authors: Klare
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D041883:Chlorobium / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010300:Parkinson Disease / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases; D020558:GTP Phosphohydrolases
keywords: G-protein; Parkinson's disease; Ras of complex proteins (Roc) domain; RocCOR tandem; Roco; conformational heterogeneity; double electron–electron resonance (DEER); electron paramagnetic resonance (EPR) spectroscopy; leucine-rich repeat kinase 2 (LRRK2); structure