Abstract
Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation – periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.
Highlights
In many Gram-negative bacteria, the periplasmic space is the major site for the maturation of proteins that enter this compartment
Phylogenetic Analysis of HP0231 To gain insight into the H. pylori Dsb system and the evolutionary origins of HP0231, we first decided to use a phylogenetic approach by performing sequence clustering analysis of the thioredoxin catalytic domains of Dsb proteins
The C-terminal catalytic domain of the HP0231 protein is localized in a tiny separate cluster, which is connected to the DsbG cluster, whereas the catalytic domain of LpDsbA2, dimeric Dsb bifunctional protein of L. pneumophila, is localized in the DsbA type-II cluster
Summary
In many Gram-negative bacteria, the periplasmic space is the major site for the maturation of proteins that enter this compartment. EcDsbA (a 21-kDa monomeric protein) directly donates its disulfide bond present in the active site to unfolded, reduced protein substrates (Paxman et al, 2009). A strictly conserved CXXC catalytic motif (CPHC in EcDsbA) present within the TRX (thioredoxin) fold is required for activity as well as a proline residue, always found in cis-conformation. The loop region containing the cis-proline residue, distant from the CXXC active site in the linear sequence but close in the three-dimensional structure, is important for maintaining both the conformation of the active site and the redox potential of the protein (Kadokura et al, 2005; Ren et al, 2009). EcDsbA activity is dependent on the action of the inner membrane protein DsbB, which restores it to the original, oxidized form. The process depends on the cell respiratory chain (Inaba, 2008)
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