Ubiquitin forms polymeric chains with eight linkage types and three shape types: linear, branched, and cyclic forms. Although linear ubiquitin chains had been thought to mainly regulate intracellular reactions in a linkage-type dependent manner, non-linear chains have recently been found to be involved in important intracellular reactions.
Linear (i.e., non-cyclic) K48-linked ubiquitin chains function in protein degradation. Although non-cyclic K48-linked ubiquitin chains are known to be cyclized by ubiquitin-conjugating enzyme E2 K in cells, the intracellular function of cyclic form remains unclear.
In this study, to elucidate the functional properties of cyclic K48-linked ubiquitin chains, by combination of NMR spectroscopic measurements with molecular dynamics (MD) simulations, we examined the difference in molecular recognition between cyclic and non-cyclic ubiquitin chains by OTUB1 that selectively cleaves K48-linked ubiquitin chains.
Real-time NMR monitoring showed that the cleavage processivity of OTUB1 for cyclic ubiquitin chains was much slower than that for non-cyclic ones. This result was reasonable because cyclization restricted the transition from the closed state to the open state in which the hydrophobic patches centered at I44 of ubiquitin moieties were accessible to OTUB1. On the other hand, we detected slow but unambiguous cleavage of cyclic ubiquitin chains by OTUB1, and we quantitatively examined that they interacted with OTUB1 weakly in spite of the cyclic conformation. In addition to the unexpected results, the low-populated alternative conformational state was detected by R2 relaxation dispersion, and coarse-grained MD simulations showed that cyclic ubiquitin chains adapt incompletely open conformations that are able to interact with binding proteins.
Taken together, our in-depth NMR spectroscopic analysis with MD simulations revealed that cyclic ubiquitin chains were recognized by intracellular proteins in a distinct manner from non-cyclic ones, and that they have the potential to exhibit characteristic intracellular functions as signal molecules.