Conformational switch upon phosphorylation: human CDK inhibitor p19INK4d between the native and partially folded state.
P19INK4d consists of five ankyrin repeats and controls the human cell cycle by inhibiting the cyclin D-dependent kinases 4 and 6. Posttranslational phosphorylation of p19INK4d has been described for Ser66 and Ser76. In the present study we show that mimicking the phosphorylation site of p19INK4d by a glutamate substitution at position 76 dramatically decreases the stability of the native but not an intermediate state. At body temperature the native conformation is completely lost and p19INK4d molecules exhibit the intermediate state as judged by kinetic and equilibrium analysis. High resolution NMR spectroscopy verified that the three C-terminal repeats remained folded in the intermediate state, whereas all cross-peaks of the two N-terminal repeats lost their native chemical shift. Molecular dynamic simulations of p19INK4d in different phosphorylation states revealed large-scale motions in phosphorylated p19INK4d, which cause destabilization of the interface between the second and third ankyrin repeat. Only doubly phosphorylated p19INK4d mimic mutants showed in vitro an increased accessibility for ubiquitination, which might be the signal for degradation in vivo.
Löw
Christian
C
Institut für Physik, Biophysik, Martin-Luther-Universität Halle-Wittenberg, D-06120 Halle (Saale), Germany.
Homeyer
Nadine
N
Weininger
Ulrich
U
Sticht
Heinrich
H
Balbach
Jochen
J
D019943:Amino Acid Substitution; D017089:Ankyrin Repeat; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D051361:Cyclin-Dependent Kinase 6 / Q000378:metabolism; D050764:Cyclin-Dependent Kinase Inhibitor p19 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D054875:Ubiquitination; D014508:Urea / Q000378:metabolism
D050764:Cyclin-Dependent Kinase Inhibitor p19; D050505:Mutant Proteins; D014508:Urea; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6