# Structural and Biochemical Characterization of Thioredoxin-2 from _Deinococcus radiotrans_
## 3 Results and Discussion
### Structural Comparison with Other Trx2 Proteins  
To our knowledge, only two full-length Trx2 structures have been deposited in the PDB database so far; one from _R. capsulatus_ (RcTrx2, PDB ID: 2 PPT) as a reduced form [23] and the other from _Yersinia pestis_ (YpTrx2, PDB ID: 3P2A, unpublished) as an oxidized form. RcTrx2 and YpTrx2 are composed of 152 and 145 amino acids and show 37% and 41% of  
Figure 5: Conserved cysteine residues in DrTrx2. A cartoon representation of the full-length structure of DrTrx2 is provided in a different orientation compared to Figure 4. The molecule is colored progressively, from blue at the N-terminus to red at the C-terminus. The amino acid residues in the active site \({}^{63}\)WCGPC67 motif, the N-terminal zinc binding site, and Asp93, which makes a hydrogen bond (dotted black line) with Trp63, are shown as sticks. Dotted rectangles indicate enlarged views of the active site \({}^{63}\)WCGPC67 motif (orange) and the N-terminal zinc binding site (blue). The zinc ion is shown as a gray sphere. The final maximum-likelihood weighted 2\(F_{\rm o}\)-\(F_{\rm c}\) electron density map of \({}^{63}\)WCGPC67 and four cysteine residues coordinated with zinc ion contoured at 1\(\sigma\) are also presented as mesh. sequence identity with DrTrx2, respectively. Multiple sequence alignment clearly showed that six cysteine residues in the N-terminal zinc binding CXXC motifs and the active site CXXC motif are well conserved among DrTrx2, RcTrx2, and YbTrx2 (Figure 6A). To compare the overall structures of the three Trx2 proteins, structures of RcTrx2 and YbTrx2 were superimposed onto that of DrTrx2. As well defined in many studies, the C-terminal Trx-fold domain of DrTrx2 is almost identical with the Trx-fold domain of RcTrx2 and YbTrx2, with r.m.s.d. values of 1.2 A for 77 C atoms and 0.9 A of 87 C\({}^{\alpha}\) atoms, respectively (Figure S5). The N-terminal zinc-finger domain of DrTrx2 also shares a similar structure with those of RcTrx2 and YbTrx2, with r.m.s.d. values of 0.5 A for 24 C\({}^{\alpha}\) atoms and 0.4 A for 22 C\({}^{\alpha}\) atoms, respectively, except for the additional 3\({}_{10}\)-helix (RcTrx2) or loop orientation (YbTrx2) followed by \(\beta\)2 (Figure 6B). While the overall structures of RcTrx2 and YbTrx2 are almost identical, with an r.m.s.d. value of 1.5 A for 123 C\({}^{\alpha}\) atoms, we found highly notable differences between the orientation of the zinc-finger domains of DrTrx2 of the three Trx2 proteins, compared to those of RcTrx2 and YbTrx2. The entire N-terminal zinc-finger domain of DrTrx2 exhibited an approximately 120\({}^{\circ}\) counterclockwise rotation compared to the position of the RcTrx2 zinc-finger domain (Figure 6C). The difference between the orientations of the N-terminal domains of the two proteins occurred at the Pro35 residue in DrTrx2. Although the adjacent Leu34 is also conserved in RcTrx2 (Leu41), two additional amino acids (Ile42, Thr43) are inserted between Leu41 and Gly44 (corresponding to Pro35 in DrTrx2) in RcTrx2. This insertion may force the N-terminal  
Figure 6: Structural comparison of DrTrx2 with other Trx2 proteins. (**A**) The structure-based sequence alignment of DrTrx2 with RcTrx2 and YbTrx2. The programs, Mullalign ([[http://multalin.toulouse.inra.fr](http://multalin.toulouse.inra.fr)]([http://multalin.toulouse.inra.fr](http://multalin.toulouse.inra.fr)), accessed on 6 March 2021) and Espript ([[http://espript.ibcp.fr](http://espript.ibcp.fr)]([http://espript.ibcp.fr](http://espript.ibcp.fr)) (accessed on 6 March 2021)), were used to visualize the alignment. White letters on red shading represent 100% identity. (**B**) Superimposition of the structures of the N-terminal zinc-finger domains from DrTrx2 (cyan), RcTrx2 (orange), and YbTrx2 (pink). The bound zinc ion for each protein is shown as a sphere. (**C**) Superimposition of the overall structures of DrTrx2 (cyan) and RcTrx2 (orange). For clarity, only DrTrx2 and RcTrx2 structures are shown.