Evolution of a dynamic molecular switch

Taylor, Susan S.; Meharena, Hiruy S.; Kornev, Alexandr P.

doi:10.1002/iub.2059

Cited by 46 publications

(59 citation statements)

References 96 publications

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“…The D2017A (DYG) mutant was not able to phosphorylate either LRRKtide or Rab8a (Figure 3) which is consistent with other kinases, since this residue is part of the regulatory triad and is crucial for the correct coordination of the Mg 2+ -ions and the γ-phosphate of ATP in the kinase active site cleft (55).…”

Section: Protein Kinase Activity Is Conserved and In Some Cases Ampsupporting

confidence: 77%

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

Schmidt

Weng²,

Aoto³

et al. 2020

Preprint

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In a multi-tiered approach, we explored how Parkinson's Disease-related mutations hijack the finely tuned activation process of Leucine-Rich Repeat Kinase 2 (LRRK2) using a construct containing the ROC, Cor, Kinase and WD40 domains (LRRK2RCKW). We hypothesized that the N-terminal domains shield the catalytic domains in an inactive state. PD mutations, type-I LRRK2 inhibitors, or physiological Rab GTPases can unleash the catalytic domains while the active kinase conformation, but not kinase activity, is essential for docking onto microtubules. Mapping solvent accessible regions of LRRK2RCKW employing hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed how inhibitor binding is sensed by the entire protein. Molecular Dynamics simulations of the kinase domain elucidated differences in conformational dynamics between wt and mutants of the DYGψ motif. While all domains contribute to regulating kinase activity and spatial distribution, the kinase domain, driven by the DYGψ motif, coordinates domain crosstalk and serves as an intrinsic hub for LRRK2 regulation.

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Section: Protein Kinase Activity Is Conserved and In Some Cases Ampsupporting

confidence: 77%

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

Schmidt

Weng²,

Aoto³

et al. 2020

Preprint

Self Cite

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show abstract

“…We measured the effect of mutating each of these residues on kinase activity. The D2017A (DYG) mutant was not able to phosphorylate either LRRKtide or Rab8a (Figure 3) which is consistent with other kinases, since this residue is part of the regulatory triad and is crucial for the correct coordination of the Mg 2+ -ions and the γ-phosphate of ATP in the kinase active site cleft (55).…”

Section: Lrrk2 Rckw Variants Spontaneously Form Laments Around Microtsupporting

confidence: 77%

Conformation and Dynamics of the Kinase Domain Drive Subcellular Location and Activation of LRRK2.

Schmidt

Weng

Aoto

et al. 2020

Preprint

Self Cite

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Background: Leucine-Rich Repeat Kinase 2 (LRRK2) is a complex multi-domain protein where LRRK2-mutations are associated with Parkinson´s Disease (PD). To explore how pathogenic PD-mutations hijack the finely tuned activation process of LRRK2, we here used a multi-tiered approach. Methods: First, the spatial and temporal distribution of full-length LRRK2 was investigated by a real-time cell-based assay in the presence and absence of LRRK2-kinase inhibitors. In a 2nd layer we explored the consequences of PD mutations as well as removal of the N-terminal domains employing a construct containing the ROC, Cor, Kinase and WD40 domains (LRRK2RCKW). We focused on the biochemical characterization of LRRK2RCKW variants based on kinase assays using Rab8a or LRRKtide as substrates. Next, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map the solvent accessible regions of LRRK2RCKW in the presence and absence of the LRRK2 inhibitor MLi-2. Finally, Molecular Dynamics simulations on the kinase domain were applied to elucidate differences in breathing dynamics between wild type and mutants of the DYGψ motif. Results: Our cellular approaches revealed that the kinase inhibitors MLi-2 and rebastinib both freeze the kinase domain in a stable conformation, however, only MLi-2 resembles an active conformation and induces filament formation. LRRK2RCKW showed, regardless of the mutation it was combined with, filament formation, indicating a shielding function of the N-terminal domains. This shielding function is impaired for pathogenic mutations in full length LRRK2. LRRK2RCKW retained kinase activity similar to full-length LRRK2. HDX-MS provided a comprehensive allosteric portrait of the kinase domain and revealed how MLi-2 binding is sensed by the entire protein. Molecular Dynamics simulations suggest that, while all domains contribute to regulating kinase activity and spatial distribution, it is the highly dynamic kinase domain, driven by the DYGψ motif, that coordinates the overall domain crosstalk and serves as a regulatory hub for the intrinsic regulation of LRRK2.Conclusion: These studies confirm our hypothesis that the N-terminal scaffolding domains shield the catalytic domains in an inactive state. PD mutations, MLi-2, or Rab GTPases can all unleash the catalytic domains while the active kinase conformation, but not kinase activity, is essential for docking onto microtubules.

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“…The αC-β4 loop is flanked by the two R-spine residues in the N-lobe, RS3, or L1924 that lies one turn of the helix beyond E1920 and RS4 or L1935 that marks the beginning of β4. RS4 is always anchored firmly to the five-stranded beta-sheet that spans the N-lobe while RS3 toggles in and out as a function of the assembly of the R-spine (Taylor et al, 2019 ). Another important interaction of the αC-β4 loop with the C-lobe is mediated by a highly conserved Tyrosine in the αE-helix.…”

Section: Conserved αC-β4 Loop Is a Hub For Structure Function And Pmentioning

confidence: 99%

Kinase Domain Is a Dynamic Hub for Driving LRRK2 Allostery

Taylor

Kaila-Sharma

Weng

et al. 2020

Front. Mol. Neurosci.

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Protein kinases and GTPases are the two major molecular switches that regulate much of biology, and both of these domains are embedded within the large multi-domain Leucine-Rich Repeat Kinase 2 (LRRK2). Mutations in LRRK2 are the most common cause of familial Parkinson’s disease (PD) and are also implicated in Crohn’s disease. The recent Cryo-Electron Microscopy (Cryo-EM) structure of the four C-terminal domains [ROC COR KIN WD40 (RCKW)] of LRRK2 includes both of the catalytic domains. Although the important allosteric N-terminal domains are missing in the Cryo-EM structure this structure allows us to not only explore the conserved features of the kinase domain, which is trapped in an inactive and open conformation but also to observe the direct allosteric cross-talk between the two domains. To define the unique features of the kinase domain and to better understand the dynamic switch mechanism that allows LRRK2 to toggle between its inactive and active conformations, we have compared the LRRK2 kinase domain to Src, BRaf, and PKA. We also compare and contrast the two canonical glycine-rich loop motifs in LRRK2 that anchor the nucleotide: the G-Loop in protein kinases that anchors ATP and the P-Loop in GTPases that anchors GTP. The RCKW structure also provides a template for the cross-talk between the kinase and GTPase domains and brings new mechanistic insights into the physiological function of LRRK2 and how the kinase domain, along with key phosphorylation sites, can serve as an allosteric hub for mediating conformational changes.

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Evolution of a dynamic molecular switch

Cited by 46 publications

References 96 publications

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

Conformation and Dynamics of the Kinase Domain Drive Subcellular Location and Activation of LRRK2.

Kinase Domain Is a Dynamic Hub for Driving LRRK2 Allostery

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