The disulfide bond isomerase DsbC is activated by an immunoglobulin-fold thiol oxidoreductase: crystal structure of the DsbC-DsbDalpha complex

Abstract

The Escherichia coli disulfide bond isomerase DsbC rearranges incorrect disulfide bonds during oxidative protein folding. It is specifically activated by the periplasmic N-terminal domain (DsbDalpha) of the transmembrane electron transporter DsbD. An intermediate of the electron transport reaction was trapped, yielding a covalent DsbC-DsbDalpha complex. The 2.3 A crystal structure of the complex shows for the first time the specific interactions between two thiol oxidoreductases. DsbDalpha is a novel thiol oxidoreductase with the active site cysteines embedded in an immunoglobulin fold. It binds into the central cleft of the V-shaped DsbC dimer, which assumes a closed conformation on complex formation. Comparison of the complex with oxidized DsbDalpha reveals major conformational changes in a cap structure that regulates the accessibility of the DsbDalpha active site. Our results explain how DsbC is selectively activated by DsbD using electrons derived from the cytoplasm.

Fig. 1. Crystal structures of the DsbC–DsbDα complex, oxidized DsbDα and DsbC C101S. (A) Structure of the DsbC–DsbDα complex. DsbDα (red) binds into the proposed substrate binding cleft of the V-shaped DsbC dimer (blue and green). The intermolecular disulfide bonds formed between the active site sulfur atoms of DsbC (Cys98) and DsbDα (Cys109) are shown as yellow spheres. DsbC assumes a closed conformation on DsbDα binding with hinge movements in both linker helices. (B) Structure of the N-terminal domain of the oxidized transmembrane electron transporter DsbD. The catalytic subdomain (blue) of DsbDα containing the active site is inserted at the antigen binding end of the Ig fold (red). The active site cysteines Cys103 and Cys109, represented by yellow spheres, form a disulfide bond in oxidized DsbDα. The active site is shielded by a cap that includes strands β6 and β7 and the loop between them. (C) The structure of the isomerase DsbC C101S in the open conformation. Each DsbC monomer consists of an N-terminal dimerization domain connected to a C-terminal catalytic domain by a linker helix. The catalytic domains have a thioredoxin fold that contains the active site cysteine residues. Figures were generated with MOLSCRIPT (Esnouf, 1999), GRASP (Nicholls et al., 1991) and RASTER3D (Merritt and Bacon, 1997). (D) Multiple sequence alignment of 10 bacterial DsbDα sequences from SwissProt (Bairoch and Apweiler, 2000) using CLUSTALX (Thompson et al., 1997). The secondary structure of DsbDα is indicated above the sequences. Conserved active site and cap residues are shown below the alignment.

Fig. 2. DsbC–DsbDα binding interactions. (A) DsbDα (red) binds into the central cleft of DsbC and contacts the catalytic domains of both monomers (blue and green). The orientation of the DsbC–DsbDα complex is that of Figure 1A. (B) Top view of the DsbC–DsbDα complex. (C) Primary and secondary binding surfaces of DsbDα. The two binding sites on DsbDα are revealed by removing DsbDα from the complex shown in (B) and rotating it by 180° around a horizontal axis. The primary binding site residues interacting with the blue DsbC monomer are shown in blue and listed on the left. The DsbDα residues of the secondary binding site are shown in green and listed on the right. (D) Primary and secondary binding surfaces of DsbC. The blue and green DsbC monomers are removed from the complex shown in (B) and rotated by approximately ±40° about a vertical axis to reveal the DsbDα binding surfaces shown in red. Residues in the primary binding site group around the active site sulfurs (yellow) of both DsbC and DsbDα. Secondary binding site residues (green and red) are located in the opposite DsbC active site and the Ig subdomain of DsbD. Figures were generated with PYMOL.

Fig. 3. Conformational changes during the thiol–disulfide exchange reaction between DsbC and DsbDα. (A) Ribbon presentation of the DsbC dimer showing the open (white) and closed (blue and green) conformation of the molecule. In the open conformation, the sulfur atoms (yellow spheres) of the two DsbC active site Cys98 are 38 Å apart. DsbC assumes a closed conformation on binding to DsbDα and the hinge movements observed in the DsbC linker helices result in the reduction of the distance between the active sites to 29 Å in the closed form. (B) Representation of the open (red) and shielded (white) form of the DsbDα active site. In the open form observed in the DsbC–DsbDα complex, the opening of the active site cap facilitates access to the DsbC binding pocket. In the shielded oxidized form of DsbDα, the binding pocket is protected from the environment by Phe70, which makes close van der Waals interactions with the active site disulfide (yellow). Phe70 moves 13 Å from its position in oxidized DsbDα.

Fig. 4. Interactions of the two DsbC active sites with DsbDα residues. (A) Stereo diagram of the primary binding site showing the DsbC active site interacting with DsbDα. The primary binding surface of DsbDα is shown colored according to the calculated electrostatic potential using GRASP. Negative charges are colored red and positive charges are in blue. Important DsbC residues (Ile96–Leu104, Gly181–Val185) are shown in blue ball-and-stick and cartoon representation. DsbC residues are labeled in black and DsbDα residues in green. DsbC Tyr100 binds into an uncharged pocket adjacent to the DsbDα active site Cys109, which forms a disulfide bond with DsbC Cys98 and hydrogen bonds to the cis-proline loop, Thr182–Pro183. (B) Stereo diagram of the secondary binding site. The electrostatic surface of the DsbDα secondary binding site is presented with DsbDα residues labeled in green. The DsbC active region is shown in green ball-and-stick and ribbons representation with yellow labels. DsbDα Asp21 interacts with the DsbC active site Cys98 and Gly99, while Tyr100 packs against DsbDα Phe22.

Fig. 5. In vivo redox state of DsbC and DsbDα derivatives. (A) Cells (dsbD^–, trxA^–) expressing the designated derivatives were induced with 0.2% arabinose, harvested at mid-log phase, precipitated with TCA and subjected to AMS treatment. Proteins were separated by SDS–PAGE and visualized by western blotting using antibodies against DsbC. DsbDα derivatives used in lanes 3–8 are mutated in amino acids in the primary binding site. The amino acids mutated in the DsbDα derivative used in lane 9 correspond to the secondary binding site. (B) As above, but using antibodies against DsbDα.