Parkinson's disease(parkinson’s disease,pd) is the second most common neurodegenerative disease, with a prevalence of 1-2% in the global older population (>65). In 2004, Andrew Singleton's group and Thomas Gasser's group discovered leucine-rich repeat kinase 2 for the first time through genetic studieslrrk2) is highly associated with Parkinson's disease, and this kinase has become one of the research hotspots in the field of Parkinson's disease。It is worth mentioning that these two scholars also won the 2024 "Oscars of Academia" for this pioneering workbreakthrough prize(Gain-of-function mutations in genes are the most common genetic factor leading to familial (5%) Parkinson's disease and a key genetic risk factor for occasional (1%) Parkinson's disease.)At present, more than 250 mutations in the LRRK2 gene have been identified, and biochemical studies have shown that some pathogenic mutants show significant enhancement of kinase activity (such as the most common pathogenic mutant LRRK2G2019S).。Therefore, LRRK2 has become a potential drug target for this disease, and the research and development of its inhibitors has aroused widespread interest among researchersIt is worth mentioning that the LRRK2-specific type I inhibitors DNL201 and DNL151 have made good progress in clinical trials
LRRK2 protein is widely expressed in organs and tissues such as brain, heart, kidney, and lung. It is involved in a variety of cellular physiological processes in vivo, including autophagy, regulation of neurite proliferation, synaptic morphogenesis, membrane trafficking, protein synthesis, and some mitochondrial diseases。The LRRK2 protein contains 2527 amino acid residues in its full length, which is composed of 7 domains, and has kinase and GTP hydrolase activities, and the assembly and mutual regulation between its internal domains are complex. Studies have shown that LRRK2 protein exists in a variety of forms of aggregation in cells, including monomers, dimers and oligomers, and the activity of LRRK2 protein in different aggregation states is also different. Some LRRK2 pathogenic mutants can form filament structures on the surface of microtubules, thereby affecting microtubule-related physiological processes. LRRK2 can be recruited and activated by the intracellular overexpressed Rab29 near the trans-Golgi network, thereby enhancing its autophosphorylation and phosphorylated Rab substrate activity and regulating downstream physiological functions。However, the molecular mechanism of LRRK2 activation and the regulation of LRRK2 protein conformation by inhibitor binding are not well understood.
December 22, 2023, St. Jude Children's Research Hospital, USASun JiResearch group and School of Life Sciences, University of Dundee, UKdario r. alessiThe research group cooperates inscienceThe magazine published a publication entitledrab29-dependent asymmetrical activation of leucine-rich repeat kinase 2Research**. The workFor the first time, the different aggregation states (monomers, dimers and tetramers) of human LRRK2 and Rab29 protein complexes and the cryo-EM structure of DNL201, a protein-binding inhibitor of LRRK2, were reported, revealing the molecular mechanism by which LRRK2 is recruited and activated by Rab29(Figure 1).
In the previous work, Sun Ji's team took the lead in reporting a series of high-resolution cryo-EM structures of human-derived full-length LRRK2, and all the LRRK2 structures resolved were in open inactive conformationAt present, the activated LRRK2 structure is limited to low-resolution microtubule-bound or type I inhibitor-bound LRRK2 structure. In order to capture the high-resolution 3D structure of activated-state LRRK2, the authors first reconstituted the RAB29-LRRK2 complex in vitro and successfully resolved its high-resolution structure, which contained three aggregation states (monomer, dimer, and tetramer). Structural display shows that Rab29GTP binds to the N-terminal ARM domain of the LRRK2 protein. Based on the analysis of structure, biochemical enzyme activity and laser confocal imaging, the interaction interface and key amino acid residues that mediate the recruitment and activation of LRRK2 by Rab29 were revealed. In the monomeric and dimer conformations, LRRK2 exhibits an open inactivation state. Surprisingly, in the tetrameric conformation of the Rab29-LRRK2 complex, the authors observed not only the peripheral LRRK2 molecule in the open inactive state (LRRK2peri), but also the intermediate LRRK2 molecule in the closed state (LRRK2cent).(Figure 1).。LRRK2CENT exhibits a series of typical characteristics of activator-state kinases, including "dyg-in", stable active loop, inward AC helix, and spatially continuous R-spine. In addition, LRRK2CENT can perfectly fit the reported cryo-ET electron density pattern of LRRK2-bound microtubules. Based on this, it is inferred that the lrrk2cent in the tetramer is in a closed active conformation. Due to the conformational changes during LRRK2 activation, some key secondary structural elements of the LRRK2cent KIN domain directly interact with the COR domain(Figure 1).to further stabilize the conformation. Mutant analysis showed that this interaction was essential for maintaining LRRK2 kinase activity.
(Figure 1).Conformational changes during LRRK2 activation.
In this paper, we further propose a model of the underlying molecular mechanism of Rab29-mediated membrane localization and asymmetric activation of LRRK2(Figure 1b).:1) Membrane-localized Rab29GTP recruits LRRK2 to form inactive monomers and dimers first, and with the increase of local LRRK2 molecular concentration, tetramers containing activated states are formed. 2) During the activation of LRRK2 (tetramer formation), LRRK2CENT undergoes a large conformational change, and its C-terminal kin-WD40 domain is close to the COR domain and directly interacts with each other, resulting in a stable closed activation conformation. 3) The self-inhibited LrRK2cent N-terminal domain (arm-ank-lrr) is released during activation and cannot be observed due to its flexible conformation. 4) The release of the N-terminal domain of LRRK2CENT leads to the exposure of the Kin domain, which can effectively exert kinase activity on membrane-localized Rab substrates. In addition, the activated state of lrrk2cent provides a good structural explanation for the working mechanism of some pathogenic mutants.
Binding of LRRK2-specific type I inhibitors can induce the formation of a closed active-like conformation of LRRK2. In order to further verify that LRRK2CENT is indeed in the closed activated state and to study the pharmacology of LRRK2, the authors resolved the cryo-EM structure of LRRK2 in combination with DNL201, a type I inhibitor. The structural comparison showed that the conformation of LRRK2 binding DNL201 was similar to that of LRRK2CENT, which once again confirmed the active conformation inference of LRRK2CENT. Finally, the authors compared and analyzed the activation of LRRK2 by Rab29 and its homologous proteins RAB32 and Rab38. Although all three can membrane localize and activate the phosphorylation activity of LRRK2 against Rab10, only the binding of Rab29 can promote the formation of LRRK2 tetramers and enhance the level of autophosphorylation at LRRK2 Ser1292(Figure 2).The level of LRRK2 Ser1292 autophosphorylation was significantly increased in the urinary exosomes of LRRK2 mutant carriers, suggesting a potential pathological association between Rab29-mediated LRRK2 tetramer formation and Ser1292 autophosphorylation. In summary,This study proposes a novel molecular mechanism of small G protein-mediated asymmetric activation of kinases, which provides a structural basis for the development and modification of LRRK2 inhibitors for Parkinson's disease**.
(Figure 2).A model of the underlying molecular mechanism by which Rab29 recruits and activates LRRK2.
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Researcher Sun Ji of St. Jude Children's Research Hospital is the corresponding author of the article. Hanwen Zhu, a postdoctoral fellow at St. Jude Children's Research Hospital, and Dr. Francesca Tonelli from the Dario Alessi Research Group, School of Life Sciences, University of Dundee, UK, are the co-first authors of this paper. In addition, Dr. Martin Turk, a staff member of the Cryo-EM Center at St. Jude Children's Research Hospital in the United States, and Dr. Alan Prescott, from the School of Life Sciences at the University of Dundee, United Kingdom, also made important contributions to this work.
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