# Structural and Biochemical Characterization of Thioredoxin-2 from _Deinococcus radiotrans_
## 3 Results and Discussion
### Structural Analysis of DrTrx2  
To further investigate the structure-function relationships, we solved the crystal structure of full length DrTrx2 at 1.96 A resolution. A native data set was collected at a wavelength of 0.97933 A. DrTrx2 crystal belongs to the \(C2_{1}\) space group with unit-cell parameters of a = 101.89 A, b = 57.82 A, and c = 36.15 A with one monomer in the asymmetric unit. The DrTrx2 structure was solved by molecular replacement, using the structure of Trx2 from \(R\). _capsulatus_ (RcTrx2, PDB ID: 2 PPT) [23] as the search model. The electron densities of all residues (Ser2-Ser142) in DrTrx2 were well defined. The final model of DrTrx2 was refined to crystallographic \(R\) and \(R_{\text{free}}\) values of 19.11% and 22.68%, respectively (Table 1). DrTrx2 is composed of a N-terminal zinc-finger domain and a C-terminal Trx-fold domain. The C-terminal Trx-fold domain is composed of a central \(\beta\)-sheet consisting of five \(\beta\)-strands and four flanking \(\alpha\)-helices on either side, where the secondary structural elements are \(\beta\)3-\(\alpha\)1-\(\beta\)4-\(\alpha\)2-\(\beta\)5-\(\alpha\)3-\(\beta\)6-\(\gamma\)4. Three \(\beta\)-strands (\(\beta\)3-\(\beta\)5) are parallel, while \(\beta\)6 and \(\beta\)7 are antiparallel. The N-terminal zinc-finger domain, coordinated with one zinc ion, consists of two \(\beta\)-sheets (\(\beta\)1 and \(\beta\)2) connected by four loops and is linked to \(\beta\)3 in the C-terminal Trx-fold domain (Figure 4A). The amino acid sequence of DrTrx2 was used to compare against 150 selected homologous sequences using the ConSurf server ([[https://consurf.tau.ac.il/](https://consurf.tau.ac.il/)]([https://consurf.tau.ac.il/](https://consurf.tau.ac.il/)), accessed on 17 April 2021) [55], to analyze the surface conservation map (Figure 4B). The results showed that the active site residues \({}^{63}\)WCGPC\({}^{67}\) in the Trx-fold domain are highly conserved and that the cysteine residues of two zinc binding CXXC motifs are also conserved at a relatively high rate, along with several conserved residues in \(\beta\)2 of the N-terminal domain. Figure 4: Overall structure of DrTrx2. (**A**) Cartoon representation of the full-length structure of DrTrx2. The molecule is colored progressively, from blue at the N-terminus to red at the C-terminus. The four \(\alpha\)-helices (\(\alpha\)1-\(\alpha\)4) and seven \(\beta\)-sheets (\(\beta\)1–\(\beta\)7) are labelled. One zinc ion bound to the N-terminal zinc-finger domain is shown as a gray sphere. (**B**) Surface conservation of the DrTrx2. The sequence conservation pattern of DrTrx2 was obtained using the Consurf server. Cartoon representation of DrTrx2 shown at the same orientation as (**A**) with a transparent surface. Residues are colored from magenta to cyan with a descending order of conservation, as shown in the color-coding bar below. The active site WCGPC motif, the zinc binding cysteines, and a zinc ion are labelled. The active site \({}^{63}\)WCGPC67 motif is located at the solvent accessible loop following \(\beta 2\) and at the beginning of \(\alpha 2\). In our DrTrx2 structure, in its oxidized state, two cysteines form a disulfide bond with a clearly connected electron density map (Figure 5). Trp63 makes a hydrogen bond with Asp93 located in the loop between \(\beta 5\) and \(\alpha 3\). This hydrogen bond was presumably participated in the stabilization of the active site configuration [56], and the same hydrogen bonds were also observed in Trx proteins from bacteria, such as _E. coli_ (PDB ID: 2 TRX) [22], _Staphylococcus aureus_ (PDB ID: 2O7K) [57], and _Mycobacterium tuberculosis_ (PDB ID: 2 IIU) [58]. The side chain of Arg68 in DrTrx2 directs towards the solvent region, while the corresponding Arg77 of RcTrx2 interacts with the backbone carbonyl group of proline residue, which precedes the conserved tryptophan residue in the WCGPC motif [23]. In the N-terminal zinc-finger domain, two CXXC motifs are located in the loops at the end of \(\beta\)-strands.