Bernd K. Gilsbach

Roco kinase structures give insights into the mechanism of Parkinson disease-related leucine-rich-repeat kinase 2 mutations.

Mutations in human leucine-rich-repeat kinase 2 (LRRK2) have been found to be the most frequent cause of late-onset Parkinson disease. Here we show that Dictyostelium discoideum Roco4 is a suitable model to study the structural and biochemical characteristics of the LRRK2 kinase and can be used for optimization of current and identification of new LRRK2 inhibitors. We have solved the structure of Roco4 kinase wild-type, Parkinson disease-related mutants G1179S and L1180T (G2019S and I2020T in LRRK2) and the structure of Roco4 kinase in complex with the LRRK2 inhibitor H1152. Taken together, our data give important insight in the LRRK2 activation mechanism and, most importantly, explain the G2019S-related increase in LRRK2 kinase activity.

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

Significance Leucine-rich repeat kinase 2 (LRRK2) represents a promising drug target for treatment and prevention of Parkinson’s disease (PD), because mutations in LRRK2 are the most common cause of Mendelian forms of the disease. PD-associated LRRK2 variants show decreased GTPase and increased kinase activity. By integrating multiple experimental inputs provided by chemical cross-linking, small-angle X-ray scattering, and a negative-stain EM map, we present, to our knowledge, the first structural model of the full-length LRRK2 dimer. The model reveals a compact folding of the LRRK2 dimer with multiple domain–domain interactions that might be involved in the regulation of LRRK2 enzymatic properties. Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson’s disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity—together with the LRR domain—to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD.

Fluoride complexes of oncogenic Ras mutants to study the Ras-RasGap interaction.

Abstract Down-regulation of Ras signalling is mediated by specific GTPase-activating proteins (GAPs), which stimulate the very slow GTPase reaction of Ras by 105-fold. The basic features of the GAP activity involve the stabilisation of both switch regions of Ras in the transition state, and the insertion of an arginine finger. In the case of oncogenic Ras mutations, the features of the active site are disturbed. To understand these features in more detail, we investigated the effects of oncogenic mutations of Ras and compared the GAP-stimulated GTPase reaction with the ability to form GAP-mediated aluminium or beryllium fluoride complexes. In general, we found a correlation between the size of the amino acid at position 12, the GTPase activity and ability to form aluminium fluoride complexes. While Gly12 is very sensitive to even the smallest possible structural change, Gly13 is much less sensitive to steric hindrance, but is sensitive to charge. Oncogenic mutants of Ras defective in the GTPase activity can however form ground-state GppNHp complexes with GAP, which can be mimicked by beryllium fluoride binding. We show that beryllium fluoride complexes are less sensitive to structural changes and report on a state close to but different from the ground state of the GAP-stimulated GTPase reaction.

Revisiting the Roco G-protein cycle

Mutations in leucine-rich-repeat kinase 2 (LRRK2) are the most frequent cause of late-onset Parkinsons disease (PD). LRRK2 belongs to the Roco family of proteins which share a conserved Ras-like G-domain (Roc) and a C-terminal of Roc (COR) domain tandem. The nucleotide state of small G-proteins is strictly controlled by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). Because of contradictory structural and biochemical data, the regulatory mechanism of the LRRK2 Roc G-domain and the RocCOR tandem is still under debate. In the present study, we solved the first nucleotide-bound Roc structure and used LRRK2 and bacterial Roco proteins to characterize the RocCOR function in more detail. Nucleotide binding induces a drastic structural change in the Roc/COR domain interface, a region strongly implicated in patients with an LRRK2 mutation. Our data confirm previous assumptions that the C-terminal subdomain of COR functions as a dimerization device. We show that the dimer formation is independent of nucleotide. The affinity for GDP/GTP is in the micromolar range, the result of which is high dissociation rates in the s-1 range. Thus Roco proteins are unlikely to need GEFs to achieve activation. Monomeric LRRK2 and Roco G-domains have a similar low GTPase activity to small G-proteins. We show that GTPase activity in bacterial Roco is stimulated by the nucleotide-dependent dimerization of the G-domain within the complex. We thus propose that the Roco proteins do not require GAPs to stimulate GTP hydrolysis but stimulate each other by one monomer completing the catalytic machinery of the other.

Conformational heterogeneity of the Roc domains in C. tepidum Roc-COR and implications for human LRRK2 Parkinson mutations

Kinetic data for leucine-rich repeat (LRR) kinase 2 (LRRK2) confirms that dimerization is essential for efficient GTP hydrolysis and that Parkinsons disease (PD) mutations cause decreased activity. Investigation of the Chlorobium tepidum RocCOR tandem reveals conformational heterogeneity of the Roc domains and the influence of LRRK2-analogous PD-mutations.

Structural Characterization of LRRK2 Inhibitors

Kinase inhibition is considered to be an important therapeutic target for LRRK2 mediated Parkinsons disease (PD). Many LRRK2 kinase inhibitors have been reported but have yet to be optimized in order to qualify as drug candidates for the treatment of the disease. In order to start a structure-function analysis of such inhibitors, we mutated the active site of Dictyostelium Roco4 kinase to resemble LRRK2. Here, we show saturation transfer difference (STD) NMR and the first cocrystal structures of two potent in vitro inhibitors, LRRK2-IN-1 and compound 19, with mutated Roco4. Our data demonstrate that this system can serve as an excellent tool for the structural characterization and optimization of LRRK2 inhibitors using X-ray crystallography and NMR spectroscopy.

Dictyostelium discoideum: A Model System to Study LRRK2-Mediated Parkinson Disease

Parkinson disease (PD) is a neurodegenerative disease that affects more than 5 million people worldwide and one in hundred people over the age of 60. PD is both a chronic and degenerative disorder that is characterized by loss of dopaminergic neurons in the substantia nigra, associated with the formation of fibrillar aggregates composed of synuclein and other proteins (Lees et al., 2009). PD is clinically characterized by tremor, bradykinesia, rigidity and postural instability. Initially PD was considered to have no genetic cause, however many patients have one or more family member with the disease and genome-wide association studies identified a number of genetic factors segregating with PD (Satake et al., 2009; Simon-Sanchez et al., 2009). Therefore, it is now general believed that PD is caused by a combination of genetic and environmental factors. Recently, missense mutations in LRRK2 have been linked to autosomal-dominant, late-onset PD (Zimprich et al., 2004;Paisan-Ruiz et al., 2004). LRRK2 is a member of the novel Roco family of complex Ras-like GTPases that have an unique domain architecture (Fig. 1) (Bosgraaf and van Haastert, 2003). Roco proteins are characterized by the presence of a Ras-like Guanine nucleotide binding domain, called Roc (Ras of complex proteins), followed by a conserved stretch of 300-400 amino-acids with no significant homology to other described protein domains called the COR domain (C-terminal of Roc; Fig. 1). The Roc and COR domains always occurs as a pair, and so far no proteins have been identified containing either the Roc or COR domain alone, suggesting that these two domains function as one inseparable unit. Roco proteins were first identified in the social amoeba Dictyostelium discoideum and are found in prokaryotes, plants and metazoa, but not in Plasmodium and yeast (Bosgraaf et al., 2003). Besides a Roc and COR domain, all Roco proteins contain an N-terminal stretch of leucine-rich repeats (LRR), which are supposed to be involved in protein-protein interaction. A large group of Roco proteins, which is only present in Dictyostelium and metazoan, contains an additional C-terminal kinase domain of the MAPKKK subfamily of kinases. Next to this general domain composition, individual Roco proteins are found to be combined with a diversity of additional domains such as Guanine nucleotide exchange factor (GEF) and Regulator of G-protein Signalling (RGS) domains, implicating a link between traditional G-protein signalling pathways and Roco proteins (Bosgraaf et al., 2003). The identification of missense mutations in LRRK2 has redefined the role of genetic variation in PD susceptibility. LRRK2 mutations initiate a penetrant phenotype with

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

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.

Regulation of LRRK2: insights from structural and biochemical analysis

Abstract Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain protein and its mutations can lead to Parkinson’s disease. Recent studies on LRRK2 and homologue proteins have advanced our mechanistic understanding of LRRK2 regulation. Here, we summarize the available data on the biochemistry and structure of LRRK2 and postulate three possible layers of regulation, translocation, monomer-dimer equilibrium and intramolecular activation of domains.