Parkinson’s disease-associated mutations in the GTPase domain of LRRK2 impair its nucleotide-dependent conformational dynamics

the mechanism involved and characteristics of this on conformation remained unknown. Here, we report that the Ras of complex protein (ROC) domain of LRRK2 exists in a dynamic dimer–monomer equilibrium that is oppositely driven by GDP and GTP binding. We also observed that the PD-associated mutations at residue 1441 impair this dynamic and shift the conformation of ROC to a GTP-bound–like monomeric conformation. Moreover, we show that residue Arg-1441 is critical for regulating the conformational dynamics of ROC. In summary, our results reveal that the PD-associated substitutions at Arg-1441 of LRRK2 alter monomer–dimer dynamics and thereby trap its GTPase domain in an activated state.

(R1441C) have been shown to have higher kinase activity (11), suggesting that mutations in the ROC domain might also up-regulate kinase activity. We previously showed that the Parkinson's diseaseassociated mutation R1441H locks the ROC domain in a prolonged activated state (12) and a recent report showed that a homologue of LRRK2, CtRoco, undergoes dimer-monomer transition upon GTP binding (13), which together suggest that perturbations in dimer-monomer dynamics might be the key factor leading to the reduction in GTPase activity observed in the PD-associated mutations at R1441.
Here, we show that all the disease-associated mutations at R1441 we examined (R1441H/G/C) showed decreased GTPase activity and a complete loss of monomer-dimer conformational dynamics. These data, taken together with prior reports, suggest that the mutations at R1441 impair monomer-dimer dynamics leading to trapping the GTPase domain of LRRK2 in a more persistently "on" state.

Arginine1441 is uniquely essential for the GTPase activity of ROC
A detailed understanding of LRRK2 has been hampered by a lack of protein samples amenable for quantitative biochemical and biophysical studies. As such, most of the detailed insights into the structure and function of LRRK2 have been gleaned from the observations of the homologous proteins. We have recently created a stably-folded G-domain construct of LRRK2 consisting of residues 1329-1520 ( Fig. 1) that we called ROC ext , which enabled us to study quantitatively the GTPase activity of wild-type ROC ext and the R1441H mutant (12). To further understand the critical effects of the disease-associated mutations and their mechanisms leading to the biochemical perturbations that we and others have observed (12,(14)(15)(16), we have created stably-folded ROC ext constructs harboring the disease-associated mutations R1441G/C (in addition to the R1441H which we have previously described), and artificial mutants R1441K/Y for detailed biochemical investigations.
We first measured the GTP hydrolysis activities of the R1441H, R1441G, and R1441C constructs and observed that the specific GTPase activity (pmol*mg -1 *min -1 ) were 9.0 ± 0.8, 12.6 ± 0.8, and 12.9 ± 0.8, respectively, which are approximately 4-fold lower than that of the wild-type (48.6 ± 0.8) (Fig. 2a). We found it notable that despite having very different sidechain properties, all the mutants showed a similar reduction in activity, which suggests that the arginine residue at position 1441 might be specifically essential for the activity of ROC ext . To test this, we made an artificial mutant substituting arginine 1441 for lysine and measured its GTPase activity. Similarly, we observed that the GTPase activity of the R1441K mutant (SA = 10.5 ± 2.5) to be about 4-fold lower than that of the wild-type (Fig. 2a). This data further supports the notion that residue arginine at position 1441 is uniquely important for ROC ext activity; thus, understanding its role would provide insights into the mechanism of this atypical G-domain and its role in disease pathogenesis.

GTPase activity and thermostability
We reported previously that the R1441H mutant has a higher affinity for GTP compared to that of WT, which we suggested might be responsible, in part, for the aberrant activity of LRRK2 (12). To determine whether or not this is a general property of mutations at R1441, we measured the affinity of the other mutants R1441G and R1441C for GDP and GTP using a fluorescence polarization (FP) assay we previously described (12). We reconfirmed that the R1441H mutant has higher affinity for GTP (K d = 3.0 ± 0.4 µM) than that of WT (K d = 4.1 ± 0.3 µM); however, they are not replicated in the R1441G and R1441C mutants, which have similar GTP affinities (K d = 4.5 ± 0.7 µM and 4.9 ± 0.8 µM, respectively) to the WT. Similarly, there are no consistent trends for GDP affinity, where the R1441C mutant has lower affinity for GDP (K d = 2.5 ± 0.4 µM) compared to that of WT (K d = 1.2 ± 0.1 µM); while the R1441G (K d = 1.8 ± 0.3 µM) and R1441H (K d = 1.9 ± 0.3 µM) binds GDP with affinities comparable to the WT (Fig. 2b). These data indicate that the higher affinity for GTP observed in the R1441H mutant is not a commonly shared property amongst the diseaseassociated mutations at position 1441; thus, it is probably not a key feature responsible for the reduction in GTPase activity observed in the mutants.
Continuing our search for the fundamental properties that rendered the R1441 mutants less active, we measured the thermal stability of each construct using a thermofluor-based protein denaturation assay (17). We observed that the R1441H ROC ext mutant is as thermally stable as the wild-type with a melting temperatures (T m ) of 54.3 ± 0.1 °C and 54.3 ± 0.1 °C, respectively (Fig. 2c); while the T m for the R1441G (T m = 50.5 ± 0.1 °C) and R1441C (T m = 51.5 ± 0.1 °C) mutants are about 3 °C lower than that of the WT (Fig.  2c). However, the GTPase activity of the R1441H mutant (despite having a higher T m ) is similar to that of R1441C and R1441G, thus it is unlikely that the lowered thermal stability of the mutants is responsible for the reduction in GTPase activity mentioned above.

The PD-associated mutations at residue 1441 disrupt dimer formation
We next examined whether the mutations at position 1441 cause structural perturbation by using circular dichroism spectroscopy (CD). We found no significant differences in the CD spectra of the mutants compared to that of the WT, indicating that the mutations have no significant impact to the secondary structure of ROC ext (Fig. 3a).
Previously we reported that the wild-type ROC ext existed in solution in a monomer-dimer equilibrium (12), which was also recently described for a LRRK2 homolog CtRoco, where the authors suggested that the disease-associated mutations in the ROC domain might disrupt the dimer-monomer dynamics (13). To investigate the effects of PDassociated mutations on the dimerization of ROC, we used multi-angle light scattering coupled to sizeexclusion chromatography (SEC-MALS). We found that all the mutants (R1441G/C/H) occurred as only monomers (Fig. 3b). This result might suggest that the monomeric form of ROC might be catalytically inactive. However, in a previous study, we showed that monomeric form of ROC is catalytically active. Moreover, we showed that the addition of GTP to the GTPase reaction renders ROC conversion from dimer to monomer. Therefore, it is more likely that the dimermonomer inter-conversion or cycling is essential for GTP hydrolysis as recently suggested for CtRoco (13).

Conformational dynamics is essential for GTPase activity
Since all the disease-associated mutations at residue R1441 abolished ROC ext dimerization, we investigated whether or not position 1441 is critical for regulating the dimer-monomer dynamics. Through systematic substitution mutations at position 1441, we found that a ROC ext containing a tyrosine residue at 1441 forms a constitutive dimer with no observable monomers (Fig.  4a). Interestingly, we found that the R1441Y dimer is catalytically inactive (Fig. 4b). A similar observation was recently made in a ROC homolog CtRoco (13), where a constitutively dimeric mutant showed reduced GTPase activity. Taken together, our data suggest that the conformational dynamics that occur in the monomer-dimer transition is essential for the GTPase activity of LRRK2 and that the disease-associated mutations at residue R1441 disrupt this activity through interfering with the dimer-monomer dynamics.
The remarkable similarities between LRRK2 ROC ext and CtRoco in their dimer-monomer dynamics suggests that their mechanism of actions might be conserved. However, a critical difference between CtRoco and our LRRK ROC ext is that CtRoco dimerization is driven by interactions in its COR domain (13,18), while our LRRK2 ROC ext construct readily forms dimers in the absence of the COR domain. This result suggests that the dimerization mechanism of LRRK2 ROC might be different from that of CtRoco. Indeed, as mentioned above, the R1441Y mutation in ROC ext locks it in a constitutive homodimeric state; however, the equivalent wild-type position in CtRoco, residue 558, is natively tyrosine. Moreover, mutations in CtRoco at Y558 mimicking the LRRK2 R1441G/C/H did not affect its dimerization, while these mutations in LRRK2 ROC ext abolished dimer formation as shown above. These similarities and differences between CtRoco and LRRK2 suggest that the subtle variability in the mechanisms of action observed in the typical Ras-and Rab-families of GTPases might also occur in the Roco family of GTPases (19).

PD-associated mutations abolish the guanine nucleotide-dependent dimer-monomer interconversions
We and others have previously shown that incubating dimeric ROC with GTP renders it monomeric (12,20), which suggest that guanine nucleotides might regulate the dynamics of ROC oligomerization. However, in our previous investigation, we observed that both GTP and GDP were equally effective for converting dimeric ROC to its monomeric conformation (12). Through further investigations, we observed that the EDTA used in the nucleotide exchange itself was sufficient to convert all the dimers into monomers. Based on this, we hypothesized that the monomerization effects of EDTA might have masked the potentially differential effects of GTP and GDP.
To investigate this, we incubated ROC ext (93% dimers) with varying concentrations of GDP or GTP without EDTA and analyzed their proportions of dimers and monomers using size-exclusion chromatography. As expected, we observed a GTP concentration-dependent conversion of dimeric ROC ext to monomers with an equilibrium proportion of dimers and monomers at 5 mM GTP to be 15% and 85%, respectively (Fig. 5a). In contrast, GDP caused a significantly smaller shift in the dimermonomer equilibrium with a ratio of dimer:monomer at 71%:29% in 5 mM GDP (Fig. 5b). These results indicate that GTP drives ROC dimers towards monomers and, reciprocally, GDP shifts the equilibrium towards the dimeric conformation. To investigate this, we developed a method to detect a dynamic conversion from dimer to monomer and then back to dimer in the same protein sample. We did so by first starting with a protein sample consisting of mostly the dimeric form of ROC ext (~85%), then we incubated it with GTP, which converts all the dimers to monomers, and then followed by exchanging it with GDP. We analyzed the dimeric state of the protein sample at each step and found that dimeric ROC ext dissociated into monomers upon GTP binding and then about 18% reassembled into dimers upon exchanging for GDP, but none converted to dimers when GTP was added instead at this stage (Fig. 5c). In contrast, the PD-associated mutation R1441G entirely abolished the dimer-monomer dynamics, where it was trapped in a monomeric conformation similar to the GTP-bound samples (Fig. 5d). These results unambiguously demonstrate a reversible dynamic interconversion between dimers and monomers that is mediated by GDP and GTP binding, respectively, and that the PDassociated mutation abolishes this dynamic process.

Conformational changes in ROC regulate LRRK2 subcellular localization
To investigate the potential significance of the observed dimer-monomer dynamics of ROC in the context of the full-length LRRK2 in live cells, we examined the subcellular localization of LRRK2 carrying the mutations that gave rise to monomeric and dimeric ROC ext . We used our previously described method to characterize interactions of LRRK2 with a binding partner RAB29(21) in HEK293FT cell. Under normal conditions LRRK2 is localized to the cytosol, but when co-transfected with RAB29 it re-localizes in clusters that overlap with the trans-Golgi network. Upon overexpression of the mutants with RAB29 in HEK293FT cells, we observed a significant increase in LRRK2 relocalization to the trans-Golgi network with all the R1441 mutations compared to WT (Fig. 6). Although the biological function of LRRK2 and its normal subcellular localization are still unclear, our results clearly demonstrate differential effects in subcellular localization between the different mutations that are shown dimeric and monomeric in our in vitro assays. Taken together, the results suggest that residue R1441 is critical for the conformational dynamics of ROC, which modulates the activity and potentially the subcellular localization of LRRK2.

Conclusion
Detailed investigations of LRRK2 has been stymied by a lack of protein samples in the quality and quantity required for quantitative studies. Recently, we reported the construction of a ROC domain and a method to obtain it in a form amenable for quantitative studies, which showed that a disease-associated mutation R1441H rendered it catalytically less active and thereby, trapping it in a more persistently "on" conformation, although we were unable to precisely define the "on conformation" at the time (12).
Here, in this report, we showed that the other diseaseassociated mutations, R1441G and R1441C, also perturb the GTPase activity of ROC, as well as abolishing their ability to form dimers. This revealed that a single substitution mutation at residue 1441 is sufficient to cause a significant conformational change in the ROC domain. We further showed that the GDPbound form of ROC exists in an equilibrium of dimers and monomers, but favoring the dimeric conformation in ratio of about 7:3; however, upon binding GTP, the ratio is shifted to 100% monomers. This indicated that GDP and GTP might regulate a cycle of dimermonomer conformational dynamics in the ROC domain of LRRK2. Indeed, we were able to demonstrate in the same protein sample that ROC ext dimers converting to monomers upon GTP binding and then cycled back to the dimeric form upon binding to GDP. This result clearly shows that the ROC ext dimer-monomer conformational change is in a dynamic equilibrium that is oppositely driven by GDP and GTP binding, and that the disease-associated mutations at residue 1441 impair this dynamic and shift the equilibrium 100% to the GTP-bound-like monomeric conformation (Fig. 7). Moreover, the results presented herein revealed that residue arginine 1441, whose mutation is associated with PD, is critical for regulating the conformational dynamics of ROC. We have recently described another PD-associated mutation in ROC, N1437H, also caused perturbation in its dimer-monomer dynamics (22), thus this might be a common effect of disease-associated mutations in the GTPase domain of LRRK2.

Size-Exclusion Chromatography Coupled with Multiangle Light Scattering (SEC-MALS)
To determine the absolute molecular weight of Roc ext in solution, we used multiple angle light scattering. Our experimental setup includes an AKTA FPLC (GE Healthcare Biosciences, Piscataway, New Jersey) with a silica-based size exclusion chromatography column (WTC-030S5, Wyatt Technology Corporation, Santa Barbara, California) as liquid chromatography (LC) unit. Down from the LC is a refractive index detector (Optilab T-rEX, Wyatt Tech.) followed by a multiple light scattering detector (Dawn HeleosII, Wyatt Tech.) for determining protein concentration and particle size, respectively. Each sample injection consisted of ~1 mg of purified ROC ext in buffer containing 30 mM HEPES (pH 7.4), 0.15 M NaCl, 10 mM MgCl 2 , 10 mM Glycine, 1 mM DTT, and 10% Glycerol. The flow rate was set at 0.4 mL/min and data were collected in a 1second interval. Data processing and analysis were performed using the ASTRA software (Wyatt Tech.)

Dimer-monomer inter-conversion assay
To determine the effect of GDP/GTP cycles on the dynamic oligomeric states, we incubated purified ROC ext with GDP or GTP in buffer containing 30 mM HEPES (pH 7.4), 0.15 M NaCl, 10 mM MgCl 2 , 10 mM Glycine, 1 mM DTT, and 10% Glycerol. This incubation was done without addition of EDTA. To test the dimeric to monomeric conversion, the dimeric protein samples (~15 mg/ml, 0.6 mM) were incubated in room temperature with 16 mM of either GDP or GTP (25× to Roc) for 6 hours. To test the monomeric to dimeric conversion, the monomeric protein samples were first obtained by GTP incubation and desalting purification (Zeba spin desalting column, Thermo Fisher). The monomeric samples were then incubated with 16 mM GDP or GTP for 6 hours. The ratios of monomeric and dimeric forms of the incubated samples were then determined by size exclusion chromatography (Superdex 75, GE Healthcare).

Circular Dichroism Spectroscopy
CD spectra were collected on a Biologic Science Instruments MOS450 AF/CD spectrometer with the slit width of 1.0 mm and data acquisition of 1.0 s. The protein samples with concentrations ranging 0.46 -0.86 mg/mL (based on absorbance at 280 nm) were dissolved in the buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, and 5% Glycerol. Data analyzed with Dichroweb and plotted with Prism 7.

GTPase Activity Assay
GTPase activity of ROC ext was assessed by using the Enzcheck assay kit (Invitrogen) according to manufacturer's instructions. Briefly, Roc ext (30 µM) was incubated with 2 mM GTP in buffer containing 30 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM MgCl 2 , 10 mM Glycine, 1 mM DTT, and 10% Glycerol at 25 ℃. Absorbance at 360 nm was recorded in every 3 minutes for 3 hours using a microplate reader. The amount of inorganic phosphate released from GTP hydrolysis at each time points was determined by extrapolation using a phosphate standard curve. Data analysis and curve fitting were done with GraphPad Prism 7.

Thermofluor Assay
Solutions of 12.5 µL of 10x Sypro Orange (prepared from 5,000x stock concentrate, Molecular Probes) in buffer containing 30 mM HEPES pH 7.4, 0.15 M NaCl, 10 mM MgCl 2 , 10 mM Glycine, 1 mM DTT, 4 mM GDP or Gpp(NH)p, 2 µM LMNG, and 10% Glycerol, and 12.5 µL of 25 µM ROC ext or mutants were added to a 96-well thin-wall PCR plate. The plate was heated in the Real-Time PCR Detection System (Mastercycler realplex, Eppendorf) from 20 to 85 ℃ and fluorescence recorded in increments of 0.4 ℃. The emission wavelength was set at 550 nm.

Subcellular localization of full-length LRRK2 mutants
Mutations in ROC were cloned into 3xFlag-LRRK2 using the Quick Change II XL Site-Directed Mutagenesis kit (Agilent Technologies). The resulting plasmids were co-transfected with 2xMyc-RAB29 into HEK293FT cells using Lipofectamine 2000 (ThermoFisher). Proteins were labeled using primary antibodies to flag (F1804 Sigma, 1:500), myc (MCA1929 BIORAD, 1:500) and TGN46 (AHP500G BIORAD, 1:1000). AlexaFluor secondary antibodies donkey anti-mouse 488, donkey anti-sheep 568, and donkey anti-rat 647 (ThermoFisher) were used at 1:500. Hoescht 33342 (H3572 ThermoFisher, 1:10000) was used as a nuclear dye. Cells were imaged, and overlap of 3xflag-LRRK2 with TGN46 was quantified using Cellomics ArrayScanVTI HCS Reader (Thermo Scientific) and HCS Studio Version 6.6.0.  GTPase activity, phosphate release against time, of wild-type (black line) ROC ext and various mutants R1441C (orange), R1441G (grey), R1441H (blue), and R1441K (green). b) Fluorescence polarization-based GDP binding assay of wild-type ROC ext (black) and the PD-associated mutants R1441C/G.H (orange, grey, and blue, respectively). Affinity shown in parenthesis. c) Fluorescence polarization-based GTP binding assay of wild-type ROC ext (black) and the PD-associated mutants R1441C/G.H (orange, grey, and blue, respectively). Affinity shown in parenthesis. d) Fluorescence-based thermal denaturation assay of ROC ext (black), R1441C (orange), R1441G (grey), and R1441H (blue). Melting temperature of shown in parenthesis.  . c) Dimer-monomer interconversion experiment showing that the same protein sample converting from mostly dimers (solid black line) to nearly completely monomers upon binding excess GTP (dashed black line) and then reverts back to forming some dimers upon binding to GDP (green dashed line), but the same treatment with GTP did not produce any dimers (red dashed line). d) The same experiment as c) for the ROC ext carrying the PD-associated mutation R1441G, showing a complete loss of nucleotide-dependent dimer-monomer interconversion.