Conformational Fluctuations Coupled to the Thiol-Disulfide Transfer between Thioredoxin and Arsenate Reductase in Bacillus subtilis*

Arsenic compounds commonly exist in nature and are toxic to nearly all kinds of life forms, which directed the evolution of enzymes in many organisms for arsenic detoxification. In bacteria, the thioredoxin-coupled arsenate reductase catalyzes the reduction of arsenate to arsenite by intramolecular thiol-disulfide cascade. The oxidized arsenate reductase ArsC is subsequently regenerated by thioredoxin through an intermolecular thiol-disulfide exchange process. The solution structure of the Bacillus subtilis thioredoxin-arsenate reductase complex represents the transiently formed intermediate during the intermolecular thiol-disulfide exchange reaction. A comparison of the complex structure with that of thioredoxin and arsenate reductase proteins in redox states showed substantial conformational changes coupled to the reaction process, with arsenate reductase, especially, adopting an “intermediate” conformation in the complex. Our current studies provide novel insights into understanding the reaction mechanisms of the thioredoxin-arsenate reductase pathway.

Thiol-disulfide exchange reactions between proteins are involved in many important cellular oxidation and reduction processes (1). Among these, the thioredoxin system, which includes NADPH, thioredoxin reductase (TrxR), 4 and thioredoxin (Trx), has been studied extensively (2)(3)(4)(5)(6). Thioredoxin is a family of small proteins (ϳ12 kDa) that exist ubiquitously in organisms from bacteria to human. Thioredoxin generally functions as protein thiol-disulfide oxidoreductase for maintaining the reducing environment in cells (2,3). Upon interaction with its substrates, the intermolecular thiol-disulfide exchange reaction is involved, and finally an intramolecular disulfide bond is formed between the two cysteine residues in Trx. The regeneration of active Trx is accomplished by TrxR, which utilizes the reducing equivalents from NADPH. Trx catalyzes the reduction of a wide range of downstream protein targets, one of which is the well documented arsenate reductase (ArsC) family (7).
ArsC catalyzes the reduction of arsenate (As(V)) to arsenite (As(III)) and is the key enzyme involved in arsenic detoxification (7). Several families of ArsC have been identified and studied extensively (8). The thioredoxin-coupled ArsC family includes Bacillus subtilis ArsC on the chromosomal skin element and the Staphylococcus aureus ArsC on the plasmid pI258. Previous studies revealed that three redox active cysteine residues (Cys 10 , Cys 82 , and Cys 89 ) are critical for the enzymatic activity of S. aureus ArsC (9), all of which are conserved in B. subtilis ArsC. A disulfide cascade mechanism was proposed based on structural, biochemical, and mutagenesis studies (10 -13). Residue Cys 10 of ArsC is responsible for the initial nucleophilic attack on arsenate to form the ArsC-As(V) covalent intermediate. A subsequent attack by Cys 82 releases the arsenite compound and introduces an intramolecular disulfide bond Cys 10 -Cys 82 . The final nucleophilic attack by Cys 89 marks the completion of a single catalytic reaction cycle, converting the enzyme into its inactive form (oxidized form) and exposing an intramolecular disulfide bridge, Cys 82 -Cys 89 , onto the protein surface. The active ArsC (reduced form) is subsequently regenerated by Trx through the intermolecular thiol-disulfide exchange reactions (14,15).
The flow of reducing equivalents through the NADPH-TrxR-Trx-ArsC-As(V) pathway involves both intra-and intermolecular thiol-disulfide exchange reactions, thereby providing a perfect model system. To date, the structures of both Trx and ArsC have been studied extensively (10 -13, 16 -18). In addition, the interaction between TrxR and Trx has also been investigated by crystal structures (4 -6). However, the transiently formed intermediate state of Trx-ArsC interaction, which would provide essential information concerning the reaction mechanism, remains poorly understood. Attempts to crystallize the Trx-ArsC complex have failed thus far (15). The only structures available for the Trx-substrate complex linked by a disulfide bond are the structures of human Trx complexed with short peptides from its substrates NFB and Ref-1 (19,20). Essential questions regarding the Trx-ArsC interaction remain unanswered: what are the transient conformations of ArsC and Trx in the complex, and how are the conformational properties of the proteins in different stages coupled to the reaction processes?
We have reported previously the solution structures and backbone dynamics of B. subtilis ArsC in both the reduced and oxidized forms (designated as re-ArsC and ox-ArsC) (13). For further understanding of the catalytic mechanisms of Trx-ArsC pathway, here we have determined the solution structures of B. subtilis Trx in both reduced and oxidized forms (designated as re-Trx and ox-Trx), and specifically we have solved the structure of the Trx-ArsC complex by NMR spectroscopy. The structure of the Trx-ArsC complex in conjunction with those of Trx and ArsC proteins in different states presents an atomic resolution view of the conformational changes along the reaction pathway.

EXPERIMENTAL PROCEDURES
Sample Preparations-The trxA gene encoding the B. subtilis Trx was cloned into the pET21a(ϩ) vector (Novagen), and the protein was expressed in Escherichia coli strain BL21(DE3). The pET28a(ϩ) arsC plasmid (13) and the pET21a(ϩ) trxA plasmid were used as DNA templates in PCR amplification with primers designed to generate mutants of ArsC_C10SC15AC82S and Trx_C32S. Sample preparations of ArsC_C10SC15AC82S, Trx and Trx_C32S were similar to that previously reported (13). The purity of the proteins was determined to be greater than 95% as judged by SDS-PAGE. The Trx-ArsC mixed disulfide complex was made using the mutants Trx_C32S and ArsC_C10SC15AC82S by the 5,5Ј-dithiobis(2-nitrobenzoic acid) incubation method following the reported protocol (15).
Structure Calculations-The details of NMR spectra collection, processing, and analysis, as well as the structure calculations of the re-Trx and ox-Trx, can be found in the supplemental "Methods" data.
The structure of Trx-ArsC complex was calculated using the program CNS (21) and refined by AMBER (22). Inter-proton NOE-derived distance restraints, the residual dipolar coupling (RDC)-derived long-range restraints, hydrogen bond restraints based on hydrogen-deuterium exchange experiments, and dihedral angle restraints based on chemical shifts (23) were used for the structure calculation. Two hundred structures were calculated using the program CNS, and the 100 structures with the lowest energy were selected and further refined using AMBER. Finally, the 20 lowest energy structures were selected as representative of the Trx-ArsC complex. The final structures were analyzed using the program packages MOLMOL (24) and PROCHECK_NMR (25).

RESULTS AND DISCUSSION
Interaction between Trx and ArsC-It has been demonstrated that the active S. aureus ArsC could be regenerated by Trx (14). To confirm that this pathway is conserved in B. subtilis, we performed in vitro experiments of the interaction between Trx and ArsC monitored by two-dimensional 15 Nedited heteronuclear single quantum coherence spectroscopy (HSQC) spectra. Unlabeled re-Trx was added into the NMR sample of the 15 N-labeled ox-ArsC, and the two-dimensional 15 N-edited HSQC spectra showed that the conformation of ArsC switched from the oxidized form to the reduced form (13). A similar experiment in which unlabeled ox-ArsC was added into 15 N-labeled re-Trx showed that the reduction of ArsC was coupled to the oxidization of Trx (supplemental Fig.  1). In contrast, we performed titration experiments using unlabeled re-Trx and 15 N-labeled re-ArsC. At a Trx:ArsC molar ratio of 1:1 or higher, the 15 N-labeled ArsC showed spectra identical to that of the re-ArsC HSQC, indicating that there is no interaction between re-Trx and re-ArsC. These results demonstrate the in vitro interaction and thiol-disulfide exchange between re-Trx and ox-ArsC. In addition, we performed in vitro enzymatic assay by measuring the oxidation of NADPH (14) and confirmed that B. subtilis ArsC activity is also coupled to the NADPH-TrxR-Trx pathway.
Solution Structure of the Trx-ArsC Complex-To fully characterize the interaction between Trx and ArsC, especially the conformational changes coupled to the redox reactions, we determined the solution structure of the B. subtilis Trx-ArsC mixed disulfide complex by NMR spectroscopy. The superimposed 20 lowest energy structures and the ribbon representation of the complex are shown in Fig. 1A. During the Trx-ArsC interaction, residue Cys 29 of Trx acts as the nucleophilic attacker and forms an intermolecular disulfide bridge with Cys 89 of ArsC. Because of the transient nature of the Trx-ArsC interaction, a stable Trx-ArsC complex with a mixed disulfide bond between Trx-Cys 29 and ArsC-Cys 89 was prepared using mutants Trx_C32S and ArsC_C10SC15AC82S following the 5,5Ј-dithiobis(2-nitrobenzoic acid) incubation procedures described by Messens et al. (15). The structure of the complex is well determined based on both NOE-derived distance restraints and RDC restraints (Table 1). A total of 112 intermolecular NOE restraints were identified at the interface of the complex, and the relative orientation of the two proteins was determined by RDC measurements. The backbone root mean square deviation from mean structure is 0.6 Ϯ 0.2 Å for the secondary structures of the whole complex and 0.23 Ϯ 0.03 and 0.39 Ϯ 0.07 Å for the individual Trx and ArsC molecules in the complex (designated as c-Trx and c-ArsC), respectively. The interaction between the two proteins buried a total of ϳ1350 Å 2 of solvent-accessible area (ϳ630 Å 2 for Trx and 720 Å 2 for ArsC), about 65% of which is contributed by nonpolar amino acid residues. The residues that are directly involved in the interaction (with unambiguous NOE peaks) include Ala 26 (Fig. 1, B-D). Conformational Changes of Trx-The detailed descriptions of structures of re-Trx and ox-Trx can be found in the supplemental data and in supplemental Fig. 2. The overall structures of re-Trx, ox-Trx, and c-Trx are similar, whereas subtle local conformational adjustments are observed in c-Trx ( Fig. 2A and  B). In particular, the short helix Gln 61 -Lys 66 in c-Trx is slightly displaced from its original positions in the free forms. This local structural change orients the helix so that its C-terminal end is shifted toward the protein surface, which can be favorable for the interactions of residues Ala 64 and Gly 65 in c-Trx with ArsC. Furthermore, residue Val 88 locates at the C-terminal end of the fifth ␤-strand in both re-Trx and ox-Trx, whereas in c-Trx it also moves away from its original position and interacts with ArsC. The conformational changes of the active site are not significant. However, the side chain of the active cysteine resi-due Cys 29 appears to adjust its positions in different states (Fig.  2B). In c-Trx, the side chain of Cys 29 moves slightly toward the protein surface to form the intermolecular disulfide bond as compared with re-Trx. After Trx switches to the oxidized form, the side chain of Cys 29 moves back and further inward because of the formation of the intramolecular Cys 29 -Cys 32 disulfide bond.
Intermediate Conformation of c-ArsC-The overall structures of ArsC in free or complex states are similar, whereas the region from residue Thr 80 to Glu 99 shows an "intermediate" conformation in c-ArsC (Fig. 2, C and D). Particularly, segment Cys 82 -Cys 89 , which is involved in a helix to loop conformational transition between re-ArsC and ox-ArsC (13), adopts an intermediate conformation in c-ArsC (Fig. 2D). The helical structure present in re-ArsC is not completely formed in the complex intermediate. However, the segment Cys 82 -Cys 89 moves a considerable distance away from its position in the ox-ArsC and shows a high tendency to forming the helix. Most residues in this segment are located at intermediate positions between the reduced and oxidized forms, but some residues show conformations closer to the reduced form. The side chain position of Ser 82 (mutated from Cys 82 ) in c-ArsC is similar to Cys 82 in re-ArsC, and the side chain of Lys 88 in c-ArsC is also much closer to its position in re-ArsC than in ox-ArsC. Residue Cys 89 , however, locates at an intermediate position in the complex and points toward the protein surface to form the intermolecular disulfide bond with Cys 29 of Trx. In contrast, the conformation of segment Pro 90 -Glu 99 appears closer to ox-ArsC (Fig. 2D). This part is mostly extended in ox-ArsC structure, but it becomes mostly coiled and moves downward (as viewed in Fig. 2, C and D) in re-ArsC. In c-ArsC, the segment Pro 90 -Glu 99 closely resembles the conformation of ox-ArsC (Fig. 2D). In addition, the segment Lys 88 -Val 96 is closer to the short helix Ser 69 -Ile 72 in re-ArsC than in ox-ArsC, whereas it is also located at an intermediate position in c-ArsC (Fig. 2C).
Biological Implications-We have presented the solution structure of the Trx-ArsC complex from B. subtilis. In the complex structure, ArsC adopts an intermediate conformation compared with the reduced and oxidized forms, representing a structural transition from the oxidized form to the reduced form. Extensive interactions between ArsC and Trx were observed in the covalently linked complex. Specifically, a pair of methionine residues were observed to insert their side chains into the other subunit. Interestingly, the E. coli TrxR-Trx interaction involved the hydrophobic side chains of a pair of arginine residues similar to that of the methionine residues in the B.
subtilis Trx-ArsC interaction (5), which may represent a coevolution of the specific residues.
In previous studies, a docking model of the S. aureus Trx-ArsC complex was proposed that placed the Cys 82 -Cys 89 loop in the interacting surface (15). Our solution structure of the B. subtilis Trx-ArsC complex, however, shows that the loop Cys 82 -Cys 89 itself does not contribute much to the Trx-ArsC interaction. Instead, the neighboring segment, Lys 88 -Pro 94 , forms most of the intermolecular interaction with Trx.
In addition, the crystal structures of the corresponding triple mutant ArsC_C10SC15AC82S of S. aureus and its 5-thio-2-nitrobenzoic acid adduct are also available (26). Both structures show a conformation similar to the reduced form of the wild type ArsC, with an intact helix between Cys 82 and Cys 89 . Because the 5-thio-2-nitrobenzoic acid adduct of S. aureus ArsC_C10SC15AC82S is able to react with Trx, it appears that the reactivity of ArsC-Cys 89 may play a major role in the reaction between ArsC and Trx.
Previous studies have suggested that S. aureus re-Trx may be able to discriminate between the folds of the reduced and the oxidized ArsC and that it interacts only with the oxidized form (15). Our results also showed that there is no interaction between the B. subtilis re-Trx and re-ArsC. Although the ability of Trx to discriminate ox-ArsC from re-ArsC based on conformational differences is still debatable, it is possible that the conformation of ox-ArsC may be preferable for interaction with Trx. In particular, the segment Lys 88 -Pro 94 undergoes extensive conformational changes during the reaction processes,  showing the most extended conformation in the oxidized state (Fig. 2, C and D). The structures of human Trx complexed with peptides of its substrates showed that the peptides adopt an extended conformation (19,20). It is reasonable to suggest that the extended conformation of segment Lys 88 -Pro 94 in ox-ArsC may facilitate its recognition and interaction by Trx. Furthermore, in re-ArsC, the closer distance between loop Lys 88 -Val 96 and helix Ser 69 -Ile 72 appears to close the groove on ArsC surface. In ox-ArsC, the loop Lys 88 -Val 96 moves away and opens up the groove, which may help to properly dock the interacting residues of both proteins into the surface grooves. It is very likely that both the reactivity of ArsC-Cys 89 and the conformation of ox-ArsC work cooperatively to facilitate the thiol-disulfide exchange reaction between Trx and ArsC in vivo. Conclusions-We have presented the solution structure of the Trx-ArsC complex, the first protein-protein complex structure between the thioredoxin and arsenate reductase families reported thus far. The structure represents an intermediate conformation during the intermolecular thiol-disulfide exchange reaction that exists only transiently under natural conditions. Our structural investigations have demonstrated the changes in the conformational properties of the enzymes at different reaction stages, which probably contribute to the progression of the reaction. The Trx-ArsC-As(V) pathway involves a series of redox reactions, and our structural and biochemical studies of the Trx-ArsC complex and the free proteins establish a dynamic picture of the conformational fluctuations of both enzymes coupled to the reaction processes.