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Figure 4
Comparison with other Trx protein complexes. The C-terminal fragment modulates the stability and redox activity of TrxT. (a) Comparison of the overall structure of TrxT with those of human Trx in complex with TXNIP (left; PDB entry 4ll1) and human Trx in complex with NFκB (right; PDB entry 1mdi), using human Trx for the fitting. The C-terminal CIVD motif of TrxT (shown in chartreuse) occupies the same position as the Trx partners in the human Trx complex structures. Full molecules are shown in Supplementary Figs. S4(a) and S4(b). (b) Model of Trx-2 and Dhd structures docked to Dm TrxR as observed in the human TrxR–Trx complex. The catalytic center of TrxR is able to access the Cys32–Cys35 bond, shown in red, in both Drosophila thioredoxins (PDB entry 3qfa). (c) Close-up view of the interaction between the catalytic center of the reductase (colored orange and indicated with an arrow) and the oxidized forms of Trx-2 (yellow) and Dhd (tan) rotated 90° with respect to the view shown in (b). The catalytic Trx and Dhd cysteines are shown in red. The α3 helix of Dm Trx and Dhd that participates in direct contacts with the reductase is labeled. (d) Close-up view of the model of TrxT bound to Dm TrxR as depicted in Fig. 4[link](b). The catalytic center of TrxR as determined in the human complex cannot access the Cys32–Cys35 bond in TrxT due to the presence of the C-terminal motif attached to Cys93 in the core domain (both sites are indicated with arrows). The orientation shown in purple will allow the reduction of Cys93–Cys125 of the tail. Once this step is achieved, a second reaction can occur to reduce the Cys32–Cys35 bond. (e) Redox activity of TrxT constructs against an eosin-labeled insulin disulfide substrate using a commercial kit developed by IMCO Corporation. The activity of the full-length TrxT protein is slightly less than that of the totally processed C-terminal domain. However, the C-terminal domain contributes to the stability of the full-length protein, increasing the melting temperature by ∼20°C. Repetitions and error bars corresponding to the standard error are indicated.

IUCrJ
Volume 8| Part 2| March 2021| Pages 281-294
ISSN: 2052-2525
https://doi.org/10.1107/S2052252521000221