Poster Presentation 23rd International Society of Magnetic Resonance Conference 2023

Structural dynamics regulate the activity of Casein Kinase 1 delta (#225)

Jakob Gebel 1 , Frank Loehr 1 , Roman Levin 1 , Charlotte Trejtnar 1 , Mahil Lambert 1 , Laura Tesmer 2 , Apirat Chaikuad 3 , Gerhard Hummer 2 , Stefan Knapp 3 , Volker Dotsch 1
  1. Institute of Biophysical Chemistry, Frankfurt University, Frankfurt am Main, Hesse, Germany
  2. Theoretical Biophysics, Max Planck Institute of Biophysics, Frankfurt am Main, Hesse, Germany
  3. Pharmaceutical Chemistry, Frankfurt University, Frankfurt am Main, Hesse, Germany

The Casein kinase 1 (CK1) family is an extremely conserved, acidophilic family of serine/threonine kinases present in virtually all eukaryotic cells. All members of the CK1 family feature a conserved substrate recognition sequence where they will phosphorylate any available S/T residue in +3 position of an already phosphorylated amino acid. They are essential for the circadian clock, Wnt signaling and DNA damage control.

Casein Kinase 1 delta (CK1δ) is involved in the circadian clock as well as a DNA damage response pathway in oocytes, which was characterized by our group. This pathway is responsible for the induction of apoptosis in oocytes with damaged genetic integrity, therefore protecting the germline1. The kinase domain of CK1δ is auto-phosphorylated at a single residue in cis, which has a substrate dependent regulatory impact on the activity of the kinase3.

TAp63a is a member of the pro-apoptotic p53 family and is expressed at a high level, but in a totally inactive form, in primordial oocytes. At least three successive phosphorylations by CK1 are required to irreversibly activate the protein. We characterized the activation kinetics by NMR and discovered a distinct ~30-fold kinetic gap between the first two phosphorylations and the decisive 3rd. We further characterized this behavior by X-ray crystallography and MD simulations. We discovered an unusual enzyme-product complex as well as a, previously unknown, secondary substrate recognition surface on the kinase2. Additionally we investigate the dynamics of substrate binding and the subsequent inhibition of the kinase by its different phosphorylation states.

By using triple-selectively labeled, cell-free expressed, CK1δ we were able to assign the majority of the protein backbone enabling us to study the dynamics of the kinase in solution with a focus on the potential novel binding pockets unveiled by crystallography and MD simulations.

  1. 1 Tuppi M, Kehrloesser S, Coutandin DW, Rossi V, Luh LM, Strubel A, Hötte K, Hoffmeister M, Schäfer B, De Oliveira T, Greten F, Stelzer EHK, Knapp S, De Felici M, Behrends C, Klinger FG & Dötsch V, Oocyte DNA damage quality control requires consecutive interplay of CHK2 and CK1 to activate p63. Nat. Struct. Mol. Biol. 25: 261–269 (2018)
  2. 2 Gebel J, Tuppi M, Chaikuad A, Hötte K, Schröder M, Schulz L, Löhr F, Gutfreund N, Finke F, Henrich E, Mezhyrova J, Lehnert R, Pampaloni F, Hummer G, Stelzer EHK, Knapp S & Dötsch V, p63 Uses a Switch-Like Mechanism To Set the Threshold for Induction of Apoptosis. Nat. Chem. Biol. 16:1078-1086 (2020)
  3. 3 Cullati, SN, Chaikuad, A, Chen, JS, Gebel, J, Tesmer, L, Zhubi, R, Navarrete-Perea, J, Guillen, RX, Gygi, SP, Hummer, G, Dötsch, V, Knapp, S, & Gould, KL, Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes. Molecular Cell, 82(11), 2006-2020.e8 (2022)