Phosphorylation at Ser65 modulates ubiquitin conformational dynamics

Structure. 2023 Jul 6;31(7):884-890.e2. doi: 10.1016/j.str.2023.05.006. Epub 2023 Jun 1.


Ubiquitin phosphorylation at Ser65 increases the population of a rare C-terminally retracted (CR) conformation. Transition between the Major and CR ubiquitin conformations is critical for promoting mitochondrial degradation. The mechanisms by which the Major and CR conformations of Ser65-phosphorylated (pSer65) ubiquitin interconvert, however, remain unresolved. Here, we perform all-atom molecular dynamics simulations using the string method with swarms of trajectories to calculate the lowest free-energy path between these two conformers. Our analysis reveals the existence of a Bent intermediate in which the C-terminal residues of the β5 strand shift to resemble the CR conformation, while pSer65 retains contacts resembling the Major conformation. This stable intermediate was reproduced in well-tempered metadynamics calculations but was less stable for a Gln2Ala mutant that disrupts contacts with pSer65. Lastly, dynamical network modeling reveals that the transition from the Major to CR conformations involves a decoupling of residues near pSer65 from the adjacent β1 strand.

Keywords: molecular dynamics; phosphorylation; string method; ubiquitin.

MeSH terms

  • Molecular Conformation
  • Molecular Dynamics Simulation*
  • Phosphorylation
  • Protein Conformation
  • Ubiquitin* / metabolism
  • Ubiquitin-Protein Ligases / chemistry


  • Ubiquitin
  • Ubiquitin-Protein Ligases