Watanabe R, Buschauer R, Böhning J, Audagnotto M, Lasker K, Lu TW, Boassa D, Taylor S, Villa E. The In Situ Structure of Parkinson's Disease-Linked LRRK2. Cell. 2020 Sep 17;182(6):1508-1518.e16. Epub 2020 Aug 11 PubMed.
Recommends
Please login to recommend the paper.
Comments
National Institute on Aging
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:
Alegre-Abarrategui J, Christian H, Lufino MM, Mutihac R, Venda LL, Ansorge O, Wade-Martins R. 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.
Dzamko N, Deak M, Hentati F, Reith AD, Prescott AR, Alessi DR, Nichols RJ. 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.
Greggio E, Jain S, Kingsbury A, Bandopadhyay R, Lewis P, Kaganovich A, van der Brug MP, Beilina A, Blackinton J, Thomas KJ, Ahmad R, Miller DW, Kesavapany S, Singleton A, Lees A, Harvey RJ, Harvey K, Cookson MR. Kinase activity is required for the toxic effects of mutant LRRK2/dardarin. Neurobiol Dis. 2006 Aug;23(2):329-41. PubMed.
Kett LR, Boassa D, Ho CC, Rideout HJ, Hu J, Terada M, Ellisman M, Dauer WT. LRRK2 Parkinson disease mutations enhance its microtubule association. Hum Mol Genet. 2012 Feb 15;21(4):890-9. Epub 2011 Nov 11 PubMed.
View all comments by Mark CooksonMedical Research Council Protein Phophorylation and Ubiquitylation Unit
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.
View all comments by Dario AlessiNational Institute on Aging
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.
View all comments by Huaibin CaiMichael J Fox Foundation for Parkinson's Research
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.
View all comments by Andrew Koemeter-CoxINSERM
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:
Ramírez MB, Ordóñez AJ, Fdez E, Madero-Pérez J, Gonnelli A, Drouyer M, Chartier-Harlin MC, Taymans JM, Bubacco L, Greggio E, Hilfiker S. GTP binding regulates cellular localization of Parkinson's disease-associated LRRK2. Hum Mol Genet. 2017 Jul 15;26(14):2747-2767. PubMed.
Dzamko N, Deak M, Hentati F, Reith AD, Prescott AR, Alessi DR, Nichols RJ. 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.
Greggio E, Zambrano I, Kaganovich A, Beilina A, Taymans JM, Daniëls V, Lewis P, Jain S, Ding J, Syed A, Thomas KJ, Baekelandt V, Cookson MR. 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.
Guaitoli G, Raimondi F, Gilsbach BK, Gómez-Llorente Y, Deyaert E, Renzi F, Li X, Schaffner A, Jagtap PK, Boldt K, von Zweydorf F, Gotthardt K, Lorimer DD, Yue Z, Burgin A, Janjic N, Sattler M, Versées W, Ueffing M, Ubarretxena-Belandia I, Kortholt A, Gloeckner CJ. 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.
Lobbestael E, Zhao J, Rudenko IN, Beylina A, Gao F, Wetter J, Beullens M, Bollen M, Cookson MR, Baekelandt V, Nichols RJ, Taymans JM. Identification of protein phosphatase 1 as a regulator of the LRRK2 phosphorylation cycle. Biochem J. 2013 Nov 15;456(1):119-28. PubMed.
Sejwal K, Chami M, Rémigy H, Vancraenenbroeck R, Sibran W, Sütterlin R, Baumgartner P, McLeod R, Chartier-Harlin MC, Baekelandt V, Stahlberg H, Taymans JM. Cryo-EM analysis of homodimeric full-length LRRK2 and LRRK1 protein complexes. Sci Rep. 2017 Aug 17;7(1):8667. PubMed.
View all comments by Jean-Marc TaymansMake a Comment
To make a comment you must login or register.