Intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase dependent mechanism

Abstract

The Parkinson's Disease (PD)-linked protein Leucine Rich Repeat Kinase 2 (LRRK2) consists of seven domains, including a kinase and a Roc G domain. Despite the availability of several high-resolution structures, the dynamic regulation of its unique intramolecular domain stack is nevertheless still not well understood. By in-depth biochemical analysis, assessing the Michaelis-Menten kinetics of the Roc G domain, we have confirmed that LRRK2 has similar to other Roco protein family members a KM value of LRRK2 that lays within the range of the physiological GTP concentrations within the cell. Furthermore, the R1441G PD variant located within a mutational hotspot in the Roc domain showed an increased catalytic efficiency. The most common PD variant G2019S, located in the kinase domain showed an increased KM and reduced catalytic efficiency, suggesting a negative feedback mechanism from the kinase domain to the G-domain. Auto-phosphorylation of the G1+2 residue (T1343) in the Roc P-loop motif is critical for this phosphoregulation of both the KM as well as the kcat values of the Roc-catalyzed GTP hydrolysis, most likely by changing the monomer-dimer equilibrium. Together our data reveal a novel intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase dependent mechanism. Interestingly, PD mutants differently change the kinetics of the GTPase cycle, which might in part explain the difference in penetrance of these mutations in PD patients.

Biophysical analysis reveals autophosphorylation as an important negative regulator of LRRK2 dimerization

“…We and others have previously shown that the Roc domain of LRRK2 is subject to autophosphorylation at multiple sites, which are in proximity to the dimerization interface within the Roc-COR module (Gloeckner et al, 2010; Greggio et al, 2009; Helton et al, 2021). Furthermore, we recently identified a novel intramolecular feedback regulation of the LRRK2 Roc G domain that depends on auto-phosphorylation of the G1+2 residue (T1343) in the Roc P-loop motif (Gilsbach et al, 2023). In contrast to wild-type, ATP dependent monomerization is abolished for T1343A, suggesting it plays a key role in regulating auto-phosphorylation dependent monomerization.…”

“…In that study, we could show that the kinetical properties of LRRK2-mediated GTP hydrolysis are negatively regulated by auto-phosphorylation (Gilsbach et al, 2023). To assess if this negative feedback is mediated via LRRK2 monomerization-dimerization, we auto-phosphorylated the purified protein under conditions allowing sufficient phospho-transfer (Gloeckner et al, 2010) and analyzed its oligomeric state.…”

Structural insights in the GTP-driven monomerization and activation of a bacterial LRRK2 homologue using allosteric nanobodies

“…PD-associated mutations in LRRK2 are concentrated in the catalytic Roc-COR and kinase domains, and commonly lead to an increase in kinase activity and - depending on the experimental set-up - a decrease in GTPase activity (Kalogeropulou et al, 2022; Lewis et al, 2007; West et al, 2005). Moreover, recent data also suggest a reciprocal cross-talk between both catalytic activities (Gilsbach et al, 2023; Störmer et al, 2023; Weng et al, 2023). Nevertheless, while the therapeutic targeting of the LRRK2 kinase domain has been a major focus of research in the last two decades, the regulation and targeting of the Roc-COR domains is only recently gaining more attention (Cogo et al, 2022; Helton et al, 2021; Park et al, 2022; Pathak et al, 2023).…”

“…Moreover, recent data also suggest a reciprocal cross-talk between both catalytic activities (Gilsbach et al, 2023;Störmer et al, 2023;Weng et al, 2023). Nevertheless, while the therapeutic targeting of the LRRK2 kinase domain has been a major focus of research in the last two decades, the regulation and targeting of the Roc-COR domains is only recently gaining more attention (Cogo et al, 2022;Helton et al, 2021;Park et al, 2022;Pathak et al, 2023).…”

Structural insights into the GTP-driven monomerization and activation of a bacterial LRRK2 homolog using allosteric nanobodies

“…PD-associated mutations in LRRK2 are concentrated in the catalytic Roc-COR and kinase domains, and commonly lead to an increase in kinase activity and -depending on the experimental set-upa decrease in GTPase activity (Kalogeropulou et al, 2022 ;Lewis et al, 2007 ;West et al, 2005 ). Moreover, recent data also suggest a reciprocal cross-talk between both catalytic activities (Gilsbach et al, 2023 ;Störmer et al, 2023 ;Weng et al, 2023 ). Nevertheless, while the therapeutic targeting of the LRRK2 kinase domain has been a major focus of research in the last two decades, the regulation and targeting of the Roc-COR domains is only recently gaining more attention (Cogo et al, 2022 ;Helton et al, 2021 ;Park et al, 2022 ;Pathak et al, 2023 ).…”