For the first time, researchers have solved the molecular structure of LRRK2, a major risk factor for Parkinson’s and autoimmune diseases. Structural biologists at the University of California, San Diego, used innovative methods to provide two complementary views of the molecule. In the August 7 Cell, researchers led by Elizabeth Villa described the three-dimensional architecture of full-length, pathogenic LRRK2 inside cells. The protein formed filaments that corkscrewed around microtubules. Researchers led by Andres Leschziner isolated a fragment of wild-type LRRK2 suitable for cryoEM. In the August 19 Nature, their high-resolution molecular maps revealed that this portion of the protein folds to bring its kinase and GTPase domains into close proximity. The finding explains previous experimental data suggesting the two domains interact.

  • By cryoimaging LRRK2, researchers solve its three-dimensional structure.
  • Kinase and GTPase domains lie close together.
  • Pathogenic mutations enhance microtubule binding, which blocks cargo transport.

By combining data from both studies, the research groups determined that for LRRK2 to bind microtubules, its kinase has to be in a closed, or active, conformation. Pathogenic mutations, some of which are known to increase microtubule binding, appear to bias the molecule toward this shape. Leschziner and colleagues’ data hint that microtubule binding could contribute to toxicity, because LRRK2 filaments impede the passage of motor proteins down these highways, potentially causing traffic jams.

LRRK2 kinase inhibitors currently being tested as PD therapeutics may also trap the protein in this closed conformation.

Microtubule Decoration. Monomers of LRRK2 (black outline) join together to form a double helix of strands (gold and blue) that wrap around microtubules (gray). Viewed down the microtubule axis, each monomer assumes the same orientation with respect to the microtubule surface (bottom). [Courtesy of Watanabe et al., Cell.]

“The new structures described in these two elegant studies represent a significant step forward in our understanding of LRRK2,” Dario Alessi at the University of Dundee, Scotland, wrote to Alzforum (full comment below). “The data provide new insights into how pathogenic mutations might exert their effects by promoting LRRK2 to adopt a closed conformation that is able to bind microtubule filaments.”

A Peek at 3-D Structures in Their Native Habitat
Mutations in LRRK2 account for up to 10 percent of familial PD cases. In addition, the concentration of this protein is elevated in many cases of sporadic disease, hinting at a broad role in pathology (Di Maio et al., 2018). Yet how LRRK2 contributes to Parkinson’s has remained murky. Besides being a kinase and GTPase, this large protein contains many other protein interaction domains (see diagram below). LRRK2 participates in numerous cellular processes, including vesicle trafficking, cell signaling, and autophagy, prompting a plethora of hypotheses about how it might cause harm (Oct 2012 news; Mar 2013 conference news). A structural map of the protein could help researchers decipher its function, but despite years of effort, LRRK2 has stubbornly resisted crystallization.

Villa and colleagues decided to flip the challenge of mapping LRRK2 on its head. Instead of trying to purify and crystallize the protein, they reasoned, why not simply image it in place? They took advantage of the fact that pathogenic LRRK2 is known to decorate microtubules, making it easy to find and visualize within the cell. Joint first authors Reika Watanabe, Robert Buschauer, and Jan Böhning expressed fluorescently tagged LRRK2 bearing the PD mutation I2020T in a kidney cell line, froze the cells, diced them into thin sections, and located LRRK2 on microtubules by correlating light and electron microscopy. Then they tilted the sections at different angles and imaged these decorated microtubules with an electron microscope in a process known as cryo-electron tomography. CryoET constructs three-dimensional structures from sequential two-dimensional images of surfaces (Lucić et al., 2008). With this technology, the authors mapped microtubule-bound LRRK2 filaments to a resolution of 14 angstroms.

Notorious Multitasker. In addition to a Ras of complex (ROC) GTPase (green), and a kinase domain (pink), LRRK2 contains many protein-protein interaction domains. The C-terminal of ROC (COR) N and C domains regulate the GTPase. These are commonly called COR-A (yellow) and COR-B (orange). [Courtesy of Watanabe et al., Cell.]

“To my knowledge, this is the first time someone has solved a structure inside a cell before it could be solved with biochemistry,” Villa said. “We took a technologically fancy but a biochemically lazy approach.”

CryoET revealed LRRK2 molecules forming a double helix around each microtubule, like the spiraling stripes of a candy cane (see image at top). In these long daisy chains of LRRK2 proteins, each bound to the one behind it through their respective WD40 domains (yes, here WD40 is a glue, not a lubricant), and to the one in front of it through their respective COR-B domains. The proteins were oriented such that their C-terminal halves, containing both catalytic domains, were located near the microtubule surface, while their N-terminal portions floated off into the cytoplasm and could not be resolved by cryoET. This orientation left the ROC GTPase domain facing the microtubule surface, and the kinase exposed to cytosol (see image below). Structural modeling suggested that the kinase was in the closed conformation, although this detail could not be resolved visually.

Dual Dimerization. Two LRRK2 monomers (left) sitting on a microtubule (outline) link via their COR domains (yellow), leaving their kinases (pink) exposed to cytosol. Their WD40 domains (red) link to adjoining monomers (gray). Rotation to show the view along the microtubule axis (right) exposes the GTPase domain (green) nestled against the microtubule surface. [Courtesy of Watanabe et al., Cell.]

Villa believes her group’s approach of using cryoET to scan molecules inside cells might help crack other recalcitrant structures, as well as provide clues to what proteins are doing in their native environment and how that changes during disease. “It’s the beginning of a new era of bridging structural and cellular biology,” Villa said.

Close Contact. The C-terminal half of LRRK2 folds to bring its kinase (orange) and GTPase (green) into proximity. Common Parkinson’s mutations are well-placed to modify this contact (right). [Courtesy of Deniston et al., Nature.]

A High-Resolution Glimpse of LRRK2’s Business End
For their part, Leschziner, co-corresponding author Samara Reck-Peterson, and colleagues took a different approach. Co-first author Sebastian Mathea in Stefan Knapp’s lab at Goethe University in Frankfurt had expressed the C-terminal half of wild-type human LRRK2 in insect cells, and found it was amenable to purification. Co-first author Colin Deniston imaged these molecules to 3.5 angstrom resolution by cryoEM. They determined that the molecule folded into a J shape that brought the ROC GTPase into close contact with the kinase domain (see image above). Previous studies had found that GTPase activity was essential for the kinase to function, but it was unclear how these domains interacted (Ito et al., 2007; West et al., 2007). In this protein fragment, the kinase assumed its open, catalytically inactive shape.

Intriguingly, the C-terminal tail of the WD40 domain formed a long α-helix that extended along the backbone of the kinase, interacting with it at several points. Noting that this α-helix contains at least one phosphorylation site, Leschziner speculated that modification of this tail might help regulate the shape of the kinase, perhaps switching it on and off.

Finally, the researchers overlaid their model onto the LRRK2 filaments described by Villa and colleagues to see if the structures matched. The monomer fit relatively well, but not perfectly—the COR domains clashed against those of the neighboring LRRK2s. When Leschziner and colleagues altered their structure to model a closed kinase domain, however, these steric clashes resolved (see image below). This finding suggested that an open conformation of the kinase would prevent microtubule binding, Leschziner said.

How Do Microtubules Fit In?
Whether LRRK2 gloms onto microtubules under physiological conditions is unclear. In cultured cells containing endogenous, wild-type LRRK2, the protein is not apparent on microtubules, Villa noted. However, when wild-type LRRK2 is overexpressed, it forms filaments on microtubules. In addition, five of the six most common PD mutations—I2020T, N1437H, R1441G, R1441C, and Y1699C—promote LRRK2 filament formation.

All of these mutations supercharge kinase activity, which would compel the kinase into its closed shape. I2020T sits in the activation loop of the kinase domain, right after G2019S, the most common pathogenic LRRK2 mutation, and the other three are at the interface between the GTPase and the COR-B domain, where they would be positioned to alter communication between the kinase and GTPase (see image above). “It seems that if you force LRRK2 into an active state, it binds microtubules,” Villa said.

Open and Shut. In the open kinase conformation (left), the LRRK2 monomer fits poorly into the filament structure. With its kinase (orange) closed (right), it clicks into place. [Courtesy of Deniston et al., Nature.]

Reck-Peterson’s data suggest that microtubule binding could cause problems. Co-first author John Salogiannis combined LRRK2, microtubules, and the motor proteins kinesin and dynein in cell-free assays. The motor proteins normally “walk” along microtubules, ferrying cargo toward the strands’ plus and minus ends, respectively. However, even low nanomolar amounts of LRRK2 shortened the distance the motors were able to walk. At 25 nM LRRK2, the motors ground to a halt, unable to step over the helical LRRK2 strands in their path.

No one knows if this roadblock serves a purpose, but Leschziner noted that LRRK2 is known to phosphorylate a subset of Rab GTPases that adorn vesicles transported along microtubules by motor proteins. Possibly, transient binding of LRRK2 oligomers to microtubules could pause motors long enough for the kinase to phosphorylate Rabs and change what cargoes get transported.

What Could This Mean for Therapy?
Questions about microtubule binding may be pertinent for PD therapy development. Type I kinase inhibitors trap the enzyme in its closed state, while keeping it inactive by preventing it from binding ATP. Do Type I LRRK2 inhibitors enhance microtubule binding? Deniston et al.’s data suggest as much, at least in cell-free assays. The researchers added the Type I inhibitor MLi-2 to their assay along with LRRK2, and found that the inhibitor further hampered motor protein movement along microtubules. MLi-2 is a pharmaceutical tool, not a drug in development (Fell et al., 2015; Scott et al, 2017). Conversely, Type II inhibitors, including the Bcr-Abl kinase inhibitor GZD-824, which stabilize an open kinase conformation, freed the motors to move again.

Mark Cookson at the National Institute on Aging, Bethesda, Maryland, noted that this finding might help explain the apparent paradox that LRRK2 kinase inhibitors enhance the formation of filaments inside cells, just as pathogenic PD mutations do. “This was particularly puzzling when considering that mutations in LRRK2 are gain-of-function, and we have thought of kinase inhibitors as potentially therapeutic,” Cookson wrote (full comment below).

“These data provide further insight into another potential pathological mechanism of LRRK2, and may explain some on-target toxicity of certain LRRK2 inhibitors, though more research is needed,” Andrew Koemeter-Cox at the Michael J. Fox Foundation wrote to Alzforum (full comment below). Likewise, Alessi suggested investigating whether Type II inhibitors would have fewer side effects.

Denali Therapeutics has two LRRK2 inhibitors, DNL201 and DNL151, in Phase 1 trials. They are both thought to be Type I. No one has yet developed LRRK2-selective Type II inhibitors, Alessi noted.

More Mysteries
The scientists are pursuing other LRRK2 riddles. Leschziner and Reck-Peterson have set their sights on the structure of other mutants. Curiously, so far G2019S has not been shown to increase LRRK2 binding to microtubules in cells. Like other pathogenic PD mutations, it turns on the kinase. However, unlike them, G2019S does not increase Rab phosphorylation in cells. “G2019S may contribute to disease in a different mechanistic way than the others,” Leschziner suggested.

To probe LRRK2’s physiological role, Villa will study endogenous LRRK2 in PD-relevant cell types, such as dopaminergic neurons and glia. In cells, the protein is more often found associated with membranes than microtubules. Does it assume a different shape when it binds membranes? Villa will recruit LRRK2 to membranes in cell culture, and combine cryoET with mass spectrometry to identify its structure and interaction partners.

Huaibin Cai at NIA believes that finding these interaction partners is crucial to deciphering what the protein does. “Future studies will be needed to determine the signaling cascades that regulate the conformational changes of LRRK2 kinase domain in different subcellular compartments, as well as to identify any particular cargoes stopped and modified by LRRK2,” he wrote to Alzforum (full comment below).—Madolyn Bowman Rogers

Comments

  1. These two papers substantially increase our structural knowledge of LRRK2, which to date has largely been based on small fragments of the protein. Identifying potential structures in situ is particularly helpful and improving the resolution with additional techniques adds insights.

    I think the most important insight is in the concept that the relative open versus closed kinase conformation may drive how the ROC-COR bi-domains are positioned. That LRRK2 can be associated with microtubules has been in the literature for a long time (Greggio et al., 2006; Kett et al., 2012) but a strange observation has been that some mutations, particularly I2020T and the GTPase deficient R1441 and Y1699 mutations, tend to show filamentous staining while kinase inhibitors also show the same (Dzamko et al., 2010). This was particularly puzzling when considering that mutations in LRRK2 are gain-of-function, and we have thought of kinase inhibitors as potentially therapeutic.

    The clarification that kinase inhibitors can evoke an open or closed conformation of the kinase domain that propagates through to GTP binding regions suggests that filament formation is only an indirect measure of what happens in the various domains of LRRK2, distinct from directly measuring kinase or GTPase function.

    One area that remains important to clarify is whether there are additional structural forms of LRRK2 in the cell, particularly those associated with cellular membranes. Again, literature from the early days of LRRK2 biology showed localization at intracellular membranes over cytoskeletal elements (Alegre-Abarrategui et al., 2009), something that has been somewhat rediscovered in recent years with the identification of Rab proteins as downstream effectors of LRRK2 function.

    The real question, therefore, is whether the microtubule association defined structurally in the current papers represents a minor, but potentially important, fraction of LRRK2 in the cell, and whether the protein in other compartments has a similar structure. It will therefore be critical to extend these techniques to examine LRRK2 associated with various membrane-bound organelles.

    References:

    . LRRK2 regulates autophagic activity and localizes to specific membrane microdomains in a novel human genomic reporter cellular model. Hum Mol Genet. 2009 Nov 1;18(21):4022-34. PubMed.

    . Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem J. 2010 Sep 15;430(3):405-13. PubMed.

    . Kinase activity is required for the toxic effects of mutant LRRK2/dardarin. Neurobiol Dis. 2006 Aug;23(2):329-41. PubMed.

    . LRRK2 Parkinson disease mutations enhance its microtubule association. Hum Mol Genet. 2012 Feb 15;21(4):890-9. Epub 2011 Nov 11 PubMed.

  2. The new structures described in these two elegant studies represent a significant step forward in our understanding of LRRK2. The data provide new insight into how pathogenic mutations might exert their effects by promoting LRRK2 to adopt a closed conformation that is able to bind microtubule filaments. The authors demonstrate convincingly in biochemical analysis that LRRK2 binding to microtubules blocks microtubule-based kinesin motors from traveling along the filaments.

    In future work, it would be important to establish that endogenous LRRK2-bearing mutations that cause Parkinson’s also bind microtubules and interfere with microtubule motility, and to address whether this is the mechanism by which LRRK2 is linked to Parkinson’s.

    To my knowledge, all widely used LRRK2 inhibitors trap LRRK2 in the closed, microtubule-binding conformation. Thus, administration of LRRK2 inhibitors that induce the closed conformation may induce LRRK2 to bind to microtubules and result in undesirable effects.

    It would therefore be interesting to develop a new class of LRRK2 inhibitors that trap LRRK2 in the open non-microtubule binding conformation. This should be possible, and a class of kinase inhibitors termed "Type-II" have been developed for other protein kinases. It would then be important to compare the impacts of treating cells with both classes of LRRK2 inhibitor, and test if administration of Type-II inhibitors that trap LRRK2 in the open non-microtubule binding conformation has reduced side effects.

  3. The interaction of LRRK2 with microtubules is among the earliest observations in the continuous endeavor to understand the biological functions of this large Parkinson's disease related multidomain protein. The memory is still vivid in my mind of our struggle to explain, in a paper published in 2009, why the presence of PD-related G2019S mutant LRRK2 proteins promotes and stabilizes microtubule assembly as opposed disrupting the microtubule network, a common pathogenic mechanism implicated in many other disease-related mutations. More than a decade later, these two elegant articles, published back-to-back by Cell and Nature, provide new structural details and functional insight into LRRK2 and microtubule co-assembly by cryo-EM and subtomogram analysis.

    Watanabe and colleagues demonstrate that LRRK2 proteins oligomerize around microtubule bundles in cultured cells. Deniston et al. further provide an atomic model of LRRK2 and microtubule association. They especially highlight the conformational changes, i.e., open/inactive versus closed/active, of the LRRK2 kinase domain in regulating the oligomerization of LRRK2 on microtubules. The closed conformation favors the association of LRRK2 polymers around microtubules, while the open conformation disfavors it.

    Moreover, they found that Type I LRRK2 kinase inhibitors promote the closed conformation, but Type II inhibitors stabilize the open structure, suggesting that different types of LRRK2 kinase inhibitor may produce confronting outcomes. In support of the functional significance of the LRRK2-microtubule interaction, Deniston et al. demonstrate that the attachment of LRRK2 polymers around microtubules blocks the kinesin and dynein motor protein-mediated cargo transport. However, LRRK2 may not act merely as a road block. For example, the wrapping of microtubules by LRRK2 may hinder the dynamic disassembly of the microtubule network and interfere with the modification of microtubule side chains.

    Future studies are needed to determine the signaling cascades that regulate the conformational changes of LRRK2 kinase domain in different subcellular compartments, as well as to identify any particular cargoes stopped and modified by LRRK2.

  4. The high-resolution structure of the RCKW domain of LRRK2 is a great leap forward in our understanding of the protein and how its domains may interact to regulate function. This data provides further insight on another potential pathological mechanism of LRRK2 and may explain some on-target toxicity of certain LRRK2 inhibitors. Though, more research is needed.

    This breakthrough gives us hope that a higher-resolution structure of LRRK2 can be achieved to aid design of improved LRRK2 inhibitors. It also validates the Michael J. Fox Foundation’s approach to addressing field-wide challenges. We funded these investigators—as we have other large collaborations—to remove a roadblock to the biological understanding of, and therapeutic development for, Parkinson’s disease.

  5. These papers by Deniston et al. and Watanabe et al. constitute significant advances in our understanding of LRRK2 structure and function. Previous insight into LRRK2 structure came through biomolecular modeling studies, three-dimensional structures of LRRK2 homologs, structures of individual domains, and, more recently, through low-resolution structures of full-length LRRK2 using TEM and cryoEM (Guaitoli et al., 2016; Sejwal et al., 2017). With these two new studies, a significant portion of the protein, including the four C-terminal domains, has now been structurally resolved with atomic resolution and in situ cryo-EM data that gives insight into the arrangement of LRRK2 C-terminal domains interacting with microtubules.

    The data provide explanations for some well-known observations, namely that LRRK2 can associate with microtubules and that LRRK2’s function is likely mediated by dynamic arrangement of its functional domains, with a key role for the kinase domain. It is an established phenomenon that some LRRK2 kinase inhibitors can induce a filamentous accumulation of LRRK2 at microtubules (Ramírez et al., 2017; Dzamko et al., 2010) and that this corresponds to changes in the LRRK2 complex, such as the loss of 14-3-3 binding, or the recruitment of phosphatases (Dzamko et al., 2010; Lobbestael et al., 2013). 

    Interestingly, this new work by Deniston and colleagues and Watanabe and colleagues now shows how LRRK2’s four C-terminal domains coordinate to decorate microtubules, and it points to differences in the induction of LRRK2 microtubule binding for type I inhibitors compared to type II inhibitors. As to function, Deniston and colleagues also show data supporting the notion that LRRK2 filament formation at microtubules constitutes a roadblock for microtubule-based motors. The implications are promising: On the one hand there is a link between LRRK2’s structure and a potentially deleterious disease-related phenotype in cells, while on the other hand there is the indication that differently designed pharmacological modulators can lead to differences in this phenotypic outcome.

    Aside from the obvious need to test the robustness of the phenotypic findings, these studies now beg further work to answer important remaining questions. If the structure-function relationship is confirmed, whereby the conformation of the kinase domain controls the ability of LRRK2’s C-terminal domains to associate with microtubules and affect traffic on the microtubule highway, then what degree of LRRK2 recruitment to microtubules could be considered healthy? Conversely, what is the precise structure (or structures) of LRRK2 when it is not associated with microtubules, and how is the transition from such a non-microtubule-associated conformation to the microtubule-associated conformation regulated under physiological and pathological conditions?

    Finally, and perhaps most importantly for our complete understanding of LRRK2’s structure, what is the structure of the three N-terminal domains, and how do these coordinate with the four C-terminal domains? The N-terminal region has been shown to affect LRRK2 biochemical activity (Greggio et al., 2008) and the previously published low-resolution Cryo-EM structure shows N-terminal domains curving away from the LRRK2 dimer core (Sejwal et al., 2017). 

    Resolving the full LRRK2 structure with the N-terminal and C-terminal domains together would answer this question and likely point to additional insights on LRRK2’s structure-function relationship in health and disease.

    References:

    . GTP binding regulates cellular localization of Parkinson's disease-associated LRRK2. Hum Mol Genet. 2017 Jul 15;26(14):2747-2767. PubMed.

    . Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem J. 2010 Sep 15;430(3):405-13. PubMed.

    . The Parkinson disease-associated leucine-rich repeat kinase 2 (LRRK2) is a dimer that undergoes intramolecular autophosphorylation. J Biol Chem. 2008 Jun 13;283(24):16906-14. Epub 2008 Apr 8 PubMed.

    . Structural model of the dimeric Parkinson's protein LRRK2 reveals a compact architecture involving distant interdomain contacts. Proc Natl Acad Sci U S A. 2016 Jul 26;113(30):E4357-66. Epub 2016 Jun 29 PubMed.

    . Identification of protein phosphatase 1 as a regulator of the LRRK2 phosphorylation cycle. Biochem J. 2013 Nov 15;456(1):119-28. PubMed.

    . Cryo-EM analysis of homodimeric full-length LRRK2 and LRRK1 protein complexes. Sci Rep. 2017 Aug 17;7(1):8667. PubMed.

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References

Alzpedia Citations

  1. Leucine-rich repeat kinase 2 (LRRK2)

News Citations

  1. The Many Faces of LRRK2
  2. LRRK Watchers’ Eyes Turn to Inflammation, Autophagy, Kinase

Therapeutics Citations

  1. DNL201
  2. DNL151

Paper Citations

  1. . LRRK2 activation in idiopathic Parkinson's disease. Sci Transl Med. 2018 Jul 25;10(451) PubMed.
  2. . Cryo-electron tomography of cells: connecting structure and function. Histochem Cell Biol. 2008 Aug;130(2):185-96. Epub 2008 Jun 20 PubMed.
  3. . GTP binding is essential to the protein kinase activity of LRRK2, a causative gene product for familial Parkinson's disease. Biochemistry. 2007 Feb 6;46(5):1380-8. PubMed.
  4. . Parkinson's disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity. Hum Mol Genet. 2007 Jan 15;16(2):223-32. PubMed.
  5. . MLi-2, a Potent, Selective, and Centrally Active Compound for Exploring the Therapeutic Potential and Safety of LRRK2 Kinase Inhibition. J Pharmacol Exp Ther. 2015 Dec;355(3):397-409. Epub 2015 Sep 25 PubMed.
  6. . Discovery of a 3-(4-Pyrimidinyl) Indazole (MLi-2), an Orally Available and Selective Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitor that Reduces Brain Kinase Activity. J Med Chem. 2017 Apr 13;60(7):2983-2992. Epub 2017 Mar 16 PubMed.

Further Reading

Primary Papers

  1. . The In Situ Structure of Parkinson's Disease-Linked LRRK2. Cell. 2020 Sep 17;182(6):1508-1518.e16. Epub 2020 Aug 11 PubMed.
  2. . Structure of LRRK2 in Parkinson's disease and model for microtubule interaction. Nature. 2020 Aug 19; PubMed.