Casein kinase 1 dynamics underlie substrate selectivity and the PER2 circadian phosphoswitch
- University of California Santa Cruz, United States
- Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore
- University of California San Diego, United States
- Duke University Medical Center, United States
Figures
Figure 1 with 1 supplement
tau alters CK1 substrate selectivity on PER2 to enhance Degron phosphorylation.
(A) Domain map of PER2 with tandem PAS domains, Casein Kinase-binding domain (CKBD), CRY-binding domain (CBD) and CK1 phosphorylation sites. (B) Sequence of the mouse PER2 FASP peptide with the …
Figure 1—figure supplement 1
The tau mutation alters substrate selectivity.
(A) Kinase assay using 20 nM CK1δ ΔC of WT or tau on 200 µM of the synthetic primed substrate, CK1tide, KRRRALpSVASLPGL (n = 4 with s.d.). (B) Phosphorylation rate of kinases on CK1tide (n = 4 with …
Figure 2 with 1 supplement
tau disrupts anion binding on CK1δ.
(A) Overlay of WT kinase domain (gray, PDB: 1CKJ, chain B) with tau (maroon, PDB: 6PXN, chain A). The 3 anion binding sites (WO42-, from 1CKJ) are labeled. (B) View of Site 1 in WT (top, gray) and ta…
Figure 2—figure supplement 1
Details of CK1 crystal structures.
(A) 2Fo-Fc omit map of the Site 1 anion binding pocket in the tau mutant (maroon, PDB: 6PXN, chain A) contoured at 1 σ. (B) View of the Site 1 anion binding pocket with WT (gray, PDB: 1CKJ, chain B) …
Figure 3 with 1 supplement
tau alters an intrinsic molecular switch in the activation loop of CK1δ.
(A) View of the activation loop switch in WT (PDB: 1CKJ, chains A (cyan) and B (gray)) and tau (PDB: 6PXN, chains A (maroon) and B (salmon)). (B) Position of L173 CD2 relative to either L152 CD2 or …
Figure 3—figure supplement 1
Crystallographic details of the CK1 activation loop switch.
(A) Overlay of the kinase domain of individual chains from tau (left, PDB: 6PXN, chains A (maroon) and B (salmon) with SO42-), WT (center, PDB: 1CKJ, chains A (cyan) and B (gray) with WO42-), and a …
Figure 4 with 1 supplement
Probing the dynamics of CK1δ with GaMD simulations.
(A-D) Stability of the activation loop assessed by the RMSD of residues 168–175 with respect to the ‘loop down’ (RMSDdown, blue) or ‘loop up’ conformation (RMSDup, pink), as observed in the crystal …
Figure 4—figure supplement 1
Additional details for GaMD simulations.
(A) Defining the localization of anion binding sites 1 (green), 2 (magenta) and 3 (yellow) for MD simulations. For reference, the loop down conformation of WT CK1δ is represented as ribbons (cyan). …
Figure 5 with 1 supplement
tau influences the principal modes of motion in CK1δ.
(A) PC1 consists of an ‘open-and-close’ movement achieved by dislocation of the N-lobe with respect to the top of the helix F to control accessibility of the ATP-binding site (dotted circle). (B) …
Figure 5—figure supplement 1
Details of the principal modes of motion.
(A) Percent of atomic fluctuations contained in each of the principal modes of motion (eigenvectors) obtained from the Principal Component Analysis of the all GaMD trajectories. The 1st and 2nd …
Figure 6 with 1 supplement
Proximity of CK1 alleles map to catalytic and substrate binding sites.
(A) Structure of CK1δ (PDB: 1CKJ, chain B) clock relevant alleles mapped from mammalian CK1 or Drosophila DBT. Mutants are colored by phenotype: short period (blue), long period (green), loss of …
Figure 6—figure supplement 1
Conservation of CK1δ/ε in eukaryotes.
(A) Alignment of the central catalytic DFG motif and activation loop of CK1ε and CK1δ (including isoforms δ1 and δ2 that differ only in the last 15 amino acids Fustin et al., 2018; Narasimamurthy et …
Figure 7 with 1 supplement
Substrate discrimination on the PER2 phosphoswitch is regulated by the CK1 activation loop switch.
(A,B) Western blot of FASP priming site, detecting pS659 (A) or Degron site, detecting pS478 (B) phosphorylation on mouse myc-PER2 in HEK293 cell lysates after transfection with indicated myc-CK1ε …
Figure 7—figure supplement 1
Effect of CK1 mutations on PER2 phosphorylation and stability.
(A) Representative real-time luminescence data for PER2::LUC stability in HEK293 cells transfected with mouse myc-PER2::LUC plus empty vector (black) or myc-CK1ε WT (gray) or mutants (red) as …
Author response image 1
Secondary structure propensity of the FASP peptide.
Videos
Video 1
Principal Component Analysis of CK1δ normal modes.
The 1st principal mode of motion corresponds to an ‘open-and-close’ movement of the kinase, achieved mainly by dislocation of the N-terminal lobe (N-lobe) with respect to the top of the helix F. The …
Additional files
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Supplementary file 1
Details of CK1 crystallography, enzyme kinetics, and simulated systems.
(A) X-ray crystallography data collection and refinement statistics. (B) Enzymatic efficiency of CK1δ ΔC (wild-type and mutants). (C) Survey of anion binding and activation loop conformation in CK1 family member structures. (D) Details of simulated systems.
- https://cdn.elifesciences.org/articles/52343/elife-52343-supp1-v1.xlsx
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Supplementary file 2
CK1 family alleles and their circadian phenotypes.
- https://cdn.elifesciences.org/articles/52343/elife-52343-supp2-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52343/elife-52343-transrepform-v1.docx
