Phosphorylation at Ser65 modulates ubiquitin conformational dynamics

SUMMARY Phosphorylation of ubiquitin at Ser65 increases the population of a rare C-terminally retracted (CR) conformation. Transition between the CR and the Major ubiquitin conformations is critical for promoting mitochondrial degradation. The mechanisms by which the Major and CR conformations of Ser65-phosphorylated (pSer65) ubiquitin interconvert, however, have not yet been revealed. 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, with the exception of a Gln2Ala mutant that disrupts contacts with pSer65. Lastly, dynamical network modelling reveals that the transition from the Major to CR conformations involves a decoupling of residues near pSer65 from the adjacent β1 strand.