Role of Dimerization in the Catalytic Properties of the Escherichia coli Disulfide Isomerase DsbC

Abstract

The bacterial protein-disulfide isomerase DsbC is a homodimeric V-shaped enzyme that consists of a dimerization domain, two ␣-helical linkers, and two opposing thioredoxin fold catalytic domains. The functional significance of the two catalytic domains of DsbC is not well understood yet. We have engineered heterodimer-like DsbC derivatives covalently linked via (Gly 3 -Ser) flexible linkers. We either inactivated one of the catalytic sites (CGYC), or entirely removed one of the catalytic domains while maintaining the putative binding area intact. Variants having a single active catalytic site display significant levels of isomerase activity. Furthermore, mDsbC[H45D]-dim[D53H], a DsbC variant lacking an entire catalytic domain but with an intact dimerization domain, also showed isomerase activity, albeit at lower levels. In addition, the absence of the catalytic domain allowed this protein to catalyze in vivo oxidation. Our results reveal that two catalytic domains in DsbC are not essential for disulfide bond isomerization and that a determining feature in isomerization is the availability of a substrate binding domain.Disulfide bonds are critical for the proper folding and structural stability of many exocytoplasmic proteins. The Dsb family of thiol:disulfide oxidoreductase enzymes catalyzes oxidative protein folding in the periplasm of Escherichia coli by means of two independent pathways (1-3). In the DsbA-DsbB oxidation pathway, DsbA, a very strong oxidant, catalyzes the formation of disulfide bonds on newly translocated proteins (4). The DsbA disulfide is rapidly recycled by DsbB, a membrane protein that transfers electrons from DsbA onto quinones (5-7). In the DsbC-DsbD isomerization pathway, non-native disulfides are reduced or rearranged by DsbC. DsbC is maintained in a reduced, catalytically active state via the transfer of electrons from the inner membrane protein DsbD that in turn accepts electrons from thioredoxin 1 and ultimately from NADPH (via thioredoxin reductase) within the cytoplasm (8, 9). Large kinetic barriers keep the oxidation and isomerization pathways isolated, preventing the establishment of a futile cycle of electron transfer. Accordingly, reactions between enzymes of the two pathways, for example the oxidation of DsbC by DsbB or the reduction of DsbA by DsbD, are 10 3 -10 7 -fold slower than the physiologically relevant DsbA-DsbB and DsbC-DsbD reactions (10). Nonetheless, the kinetic barrier between DsbB and DsbC can be breached by introducing mutations that result in structural changes in DsbC (11,12).DsbC is a homodimer with each monomer comprising an N-terminal dimerization domain and a C-terminal thioredoxin-like catalytic domain fused by an ␣-helical linker. The crystal structure of DsbC reveals that the two monomers come together to form a V-shaped protein. The inner surface of the resulting cleft is patched with uncharged and hydrophobic residues suggesting an important role in the binding of substrate proteins. The active sites comprising the sequence Cys 98 -Gly 99 -Tyr 100 -...

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox‐dependent oligomeric modulation in vitro

“…[25]. This suggests that XfDsbC Red is a dimer in solution, similar to other DsbC proteins [18,26]. The XfDsbC Red distance distribution function, P(r), showed a maximum intramolecular distance (D max ) of 100 Å (Fig.…”

“…The dimerization domains of EcDsbC and HiDsbC are primarily connected by hydrogen-bond interactions formed by main chain residues from two external antiparallel b-strands. An ionic interaction between EcDsbC His45 and Asp53 also contributes to the stability of the dimer and protein isomerase activity [26,29]. Therefore, the conservation of this characteristic interface in the XfDsbC structure was investigated.…”

“…An arginine (Arg68) and an aspartate (Asp77) residue are located on the extremities of this b-strand. Because Arg and Asp are oppositely charged, it is possible that ionic interactions help to stabilize the XfDsbC dimer, as observed in EcDsbC [26,29]. Based on this potential XfDsbC dimeric interface, the XfDsbC dimer was modelled according to the experimental XfDsbC low-resolution scattering curve.…”

“…A second XfDsbC chain was generated and superimposed on the remaining EcDsbC monomer. The putative XfDsbC dimer interface was proposed based on the high-resolution structures, chimeric constructs and site-directed mutagenesis of other DsbC dimeric proteins [18,[26][27][28]. The dimerization domains of EcDsbC and HiDsbC are primarily connected by hydrogen-bond interactions formed by main chain residues from two external antiparallel b-strands.…”

The Multidrug Resistance IncA/C Transferable Plasmid Encodes a Novel Domain-swapped Dimeric Protein-disulfide Isomerase

“…5C) are proposed to be involved in substrate recognition (16,57). By analogy, we hypothesize that the inverted V-shaped DsbP also uses the "bottom" surface of the dimerization domain for substrate recognition.…”