Biology subjects

Galicia, C.

Publications and source records attributed to Galicia, C..

5 recordsLinked to original sources

bioRxiv2026

The phosphorylation status of LRRK2 at the S910/S935 cluster determines its sensitivity to activation by RAB29

Leucine-Rich Repeat Kinase 2 (LRRK2) is a signaling molecule involved in Parkinsons disease pathomechanisms. In disease, the LRRK2 protein displays both a toxic gain of kinase function and a loss of phosphorylation at heterophosphosites found in an extended loop of the LRR domain. RAB GTPases, such as RAB29, have been identified as upstream activators of LRRK2. Indeed, co-expression of LRRK2 with RAB29 induces a hyperactivation of LRRK2 kinase activity, however the role of the LRRK2 heterologous phosphorylation status in its activation remains unknown. Here, our aim was to determine the role of LRRK2 heterologous phosphorylation on its activation by RAB29. Using single and compound phosphodead or phosphomimetic mutants of LRRK2 we show differential sensitivity of LRRK2 phosphomutants to activation by RAB29, with phosphodead mutants being more susceptible to be activated than phosphomimetic mutants. Interestingly, we find that the single phosphodead S910A LRRK2 mutant displays an activation of LRRK2 kinase activity similar to that observed for the compound phosphodead 6xS>A LRRK2 mutant (S860A/S910A/S935A/S955A/S973A/S976A). Time-course analysis revealed that phosphodead mutants displayed higher but also faster activation by RAB29. In addition, both physical interaction between LRRK2 and RAB29 as well as RAB29-induced recruitment of LRRK2 to the trans-Golgi network (TGN) was enhanced by phosphodead compared to phosphomimetic mutants. To confirm effects on native LRRK2, we tested a panel of ten nanobodies targeting LRRK2 that stabilized LRRK2 phosphorylation at varying levels. Nanobodies stabilizing LRRK2 at low S935 phosphorylation levels showed enhanced RAB29-induced activation compared to nanobodies not affecting pS935 LRRK2. Finally, we tested whether LRRK2 heterologous phosphorylation could affect centrosome cohesion deficits, a phenotype that has been linked to LRRK2 hyperactivation, and found that both the phosphodead LRRK2 as well as a nanobody stabilizing dephosphorylated LRRK2 enhanced the centrosome cohesion deficit. Our findings indicate that hyperactivability of LRRK2 is directly related to its heterologous phosphorylation status, with dephosphorylation leading to strong hyperactivation of LRRK2 by upstream activating RABs, and phosphorylated LRRK2 showing the opposite. This implies that strategies favoring LRRK2 phosphorylation will have therapeutic benefit.

neuroscience↗

bioRxiv2024

Cryo-EM structures of the MnmE-MnmG complex reveal large conformational changes and provide new insights into the mechanism of tRNA modification

MnmE and MnmG form a conserved protein complex responsible for the addition of a 5-carboxymethylaminomethyl (cmnm5) group onto the wobble uridine of several tRNAs. Within this complex, both proteins collaborate intensively to catalyze a tRNA modification reaction that involves glycine as a substrate in addition to three different cofactors, with FAD and NADH binding to MnmG and methylenetetrahydrofolate (5,10-CH2-THF) to MnmE. Without structures of the MnmEG complex it remained enigmatic how these substrates and co-factors can be brought together in a concerted manner. Prior SAXS data suggested that the MnmE (2) and MnmG ({beta}2) homodimers can adopt either an 2{beta}2 or 4{beta}2 complex, depending on the nucleotide state of MnmE. Here, we report the cryo-EM structures of the MnmEG complex in the 2{beta}2 and 4{beta}2 oligomeric states. These structures reveal that MnmE undergoes large conformational changes upon interaction with MnmG, resulting in an asymmetric MnmE dimer. In particular, the functionally important C-terminal helix of MnmE relocates from the 5,10-CH2-THF binding pocket of MnmE to the FAD binding pocket of MnmG, thus suggesting a mechanism for the transfer of an activated methylene group from one active site to the other. Together, these findings provide crucial new insights into the MnmEG-catalyzed reaction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/625835v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@144e41forg.highwire.dtl.DTLVardef@1dce537org.highwire.dtl.DTLVardef@14c9359org.highwire.dtl.DTLVardef@5dec04_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Development of LRRK2 designed ankyrin-repeat proteins

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinsons disease (PD) has led to intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its activity are needed. Here, we identified a series of high-affinity LRRK2-binding designed ankyrin-repeat proteins (DARPins). One of these DARPins (E11) bound to the LRRK2 WD40 domain with high affinity. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

biochemistry↗

bioRxiv2023

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

The Roco proteins are a family of GTPases, characterized by the conserved presence of a Roc-COR tandem domain. These proteins entered the limelight after mutations in human LRRK2 were identified as a major cause of familial Parkinsons disease. LRRK2 is a large and complex protein combining a GTPase and protein kinase activity, and disease mutations increase the kinase activity, while presumably decreasing the GTPase activity. Although a cross-communication between both catalytic activities has been suggested, the underlying mechanisms and the regulatory role of the GTPase domain remain unknown. Recently, several structures of LRRK2 have been reported, but so far structures of Roco proteins in their activated GTP-bound state are lacking. Here, we use single particle cryo-EM to solve the structure of a simpler bacterial Roco protein (CtRoco) in its GTP-bound state, aided by the use of two conformation-specific nanobodies: NbRoco1 and NbRoco2. This structure presents CtRoco in an active monomeric state, featuring very significant conformational changes compared to the previously solved nucleotide-free dimer structure. In particular, the structure shows a very large GTP-induced conformational change of the LRR domain, unleashing it from the Roc-COR domains, using the LRR-Roc linker as a hinge. Furthermore, this structure shows how NbRoco1 and NbRoco2 collaborate to activate CtRoco in an allosteric way. Altogether, our data provide important new insights in the activation mechanism of Roco proteins, with relevance to LRRK2 regulation, and suggest new routes for the allosteric modulation of their GTPase activity.

biochemistry↗

bioRxiv2019

MglA functions as a three-state GTPase to control movement reversals of Myxococcus xanthus

In Myxococcus xanthus, directed movement is controlled by inter-dependent pole-to-pole oscillations of the small GTPase MglA, its GAP MglB and the RomR protein. However, these proteins have strikingly different oscillatory regimes such that MglA is segregated from MglB and RomR at reversal activation. The molecular mechanism whereby information is exchanged between the lagging and leading poles resulting in MglA detachment from the leading pole during reversals has remained unknown. Here, we show that MglA has two GTP-bound forms, one of which is insensitive to MglB (MglA-GTP*) and is re-sensitized to MglB by a feedback mechanism operated by MglA-GDP. By identifying the region of MglB that is critical for its association to the lagging pole, we demonstrate that MglA-GTP* is functional in vivo. These data suggest that MglA-GDP acts as a soluble messenger to convert polar MglA-GTP* into a diffusible MglA-GTP species, explaining MglA re-localization to the opposite pole during reversals.

microbiology↗