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