Extended Data Fig. 8: TXNL1 dissociates from proteasomes as they transition back from processing to the resting state, but remains bound when degradation is stalled.
From: Structural landscape of the degrading 26S proteasome reveals conformation-specific binding of TXNL1
a) Left: TXNL1’s PITH domain is necessary and sufficient for the conformational selective binding to actively degrading 26S proteasomes. Shown are representative traces for the fluorescence polarization of FAM-labeled TRX domain (50 nM) or PITH domain (50 nM) that was incubated in isolation or with human 26S proteasome (500 nM) in the presence of 10 μM FAT10 substrate and 12 μM NUB1 cofactor. Right: Proteasomes whose proteolytic cleavage of the FAT10 substrate was inhibited by MG132 and which were therefore stalled during substrate translocation interact stably with TXNL1. Shown are the fluorescence polarization time courses for N-terminally FAM-labeled full-length FAMTXNL1 (50 nM) that was incubated with FAT10 (5 μM), NUB1 (6 μM), and human 26S proteasome (500 nM) in the absence or presence of MG132 (20 μM), as indicated. b) Titration of proteasomes that were stalled during substrate degradation through MG132 inhibition of proteolysis reveals the TXNL1 binding affinity for processing-state proteasomes. Left: Shown are the fluorescence polarization traces for FAMTXNL1 (50 nM) incubated with human 26S proteasome at indicated concentrations in the presence of FAT10 substrate (5 μM) and NUB1 cofactor (6 μM). Right: FAMTXNL1 binding curve derived from the 900 s time points of the polarization traces shown on the left indicates an affinity of KD ~ 35 nM.
