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Kinetic Constraints in the Specific Interaction between Phosphorylated Ubiquitin and Proteasomal Shuttle Factors.

ABSTRACT: Ubiquitin (Ub) specifically interacts with the Ub-associating domain (UBA) in a proteasomal shuttle factor, while the latter is involved in either proteasomal targeting or self-assembly coacervation. PINK1 phosphorylates Ub at S65 and makes Ub alternate between C-terminally relaxed (pUb_RL) and retracted conformations (pUb_RT). Using NMR spectroscopy, we show that pUb_RL but not pUb_RT preferentially interacts with the UBA from two proteasomal shuttle factors Ubqln2 and Rad23A. Yet discriminatorily, Ubqln2-UBA binds to pUb more tightly than Rad23A does and selectively enriches pUb_RL upon complex formation. Further, we determine the solution structure of the complex between Ubqln2-UBA and pUb_RL and uncover the thermodynamic basis for the stronger interaction. NMR kinetics analysis at different timescales further suggests an indued-fit binding mechanism for pUb-UBA interaction. Notably, at a relatively low saturation level, the dissociation rate of the UBA-pUb_RL complex is comparable with the exchange rate between pUb_RL and pUb_RT. Thus, a kinetic constraint would dictate the interaction between Ub and UBA, thus fine-tuning the functional state of the proteasomal shuttle factors.

SUBMITTER: Qin LY

PROVIDER: S-EPMC8301994 | biostudies-literature |

REPOSITORIES: biostudies-literature

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