Structure of C-terminally truncated ASK1.

(a) Schematic domain structure of ASK1. TBD, thioredoxin-binding domain; TPR, tetratricopeptide repeats; PH, pleckstrin-homology domain; CRR, central regulatory region; KD, kinase domain; CC, coiled-coil motif; SAM, sterile alpha motif domain. Black bar represents construct used in cryo-EM analysis. (b, c) Cryo-EM density map and cartoon view of C-terminally truncated dimeric ASK1. Density maps were generated using threshold level 5.5.

All four N-terminal domains of ASK1 are involved in extensive interdomain and interchain interactions.

(a) Cartoon representation of TBDA and its interaction with helix α15 of TPRB. The dashed line indicates the missing section (residues 228-245). (b) Cartoon representation of interactions between KDB, TPRA and PHA domains (shown in red, violet and orange, respectively). The activation segment is shown in green. (c) Detailed view of the active site of KDB. KDB is shown in red, TPRA is shown in violet. The activation segment is shown in green.

TBD-CRR forms dimers in solution, but not TBD or CRR.

(a-f) Sedimentation coefficient distributions (c(s)) of different ASK1 N-terminal constructs (TBD, CRR, KD, TBD-CRR, CRR-KD and TBD-CRR-KD) measured at concentrations of 4 μM, 12 μM and 20 μM. Schematic domain structure of ASK1 is shown at the top; the black bar represents the used construct. TBD, thioredoxin-binding domain; TPR, tetratricopeptide repeats; PH, pleckstrin-homology domain; CRR, central regulatory region; KD, kinase domain; SAM, sterile alpha motif domain.

TRX1 binding induces structural changes in all N-terminal domains of ASK1.

(a) Differences in ASK1 TBD-CRR-KD deuteration with and without TRX1 after 600 s. Positive values indicate protection (lower deuterium uptake) after TRX1 binding. The graph shows the average of three replicates (black points) and the standard deviation (light orange). Grey zones indicate areas without coverage. The domain structure of ASK1 TBD-CRR-KD is shown at the top. TBD, thioredoxin-binding domain; TPR, tetratricopeptide repeats; PH, pleckstrin-homology domain; CRR, central regulatory region; KD, kinase domain. (b) Cartoon representation of the structure of the ASK1 TBD-CRR-KD dimer (chain A in white, chain B in light yellow) colored according to changes in deuteration in the presence of TRX1 after 600 s. Changes in deuteration greater than twice the standard deviation and above 3% were considered significant. The insets show detailed views of the TBDA and its interface with TPRB (blue box), CRRA (orange box) and KD dimer (brown box). (c) Differences in TRX1 deuteration with and without ASK1 TBD-CRR-KD after 600 s. Positive values indicate protection (lower deuterium uptake) after ASK1 TBD-CRR-KD binding. The graph shows the average of three replicates (black points) and the standard deviation (light orange). The secondary structure of TRX1 is shown at the top. (d) Cartoon representation of TRX1 structure (in reduced state, PDB ID: 1ERT52) colored according to changes in deuteration after ASK1 TBD-CRR-KD binding for 600 s.

Proposed schematic model of ASK1 activation.

In the resting state, ASK1 constitutively oligomerizes and is kept in an inactive state through interactions with TRX1 and 14-3-3 proteins. TRX1 appears to function as an allosteric effector whose binding affects the structure of TBD, likely affecting its interaction with TPR. Therefore, TRX1 affects the structure of CRR and allosterically modulates several regions of KD, even reducing access to the activation segment with the key phosphorylation site T838. Oxidative stress triggers TRX1 dissociation, followed by 14-3-3 dissociation and TRAF recruitment. These events subsequently lead to a conformational change in KD and increase access to the activation segment, enabling its phosphorylation at T838 and, as a result, stabilizing the active conformation. The role of 14-3-3 proteins in ASK1 inhibition and the mechanism whereby TRAFs and Daxx are involved in ASK1 activation remain unclear and should be explored in subsequent studies.