Abstract
The formation of disulfide bonds that are catalyzed by proteins of the Dsb (disulfide bond) family is crucial for the correct folding of many extracytoplasmic proteins. Thus, this formation plays an essential, pivotal role in the assembly of many virulence factors. The Helicobacter pylori disulfide bond-forming system is uncomplicated compared to the best-characterized Escherichia coli Dsb pathways. It possesses only two extracytoplasmic Dsb proteins named HP0377 and HP0231. As previously shown, HP0377 is a reductase involved in the process of cytochrome c maturation. Additionally, it also possesses disulfide isomerase activity. HP0231 was the first periplasmic dimeric oxidoreductase involved in disulfide generation to be described. Although HP0231 function is critical for oxidative protein folding, its structure resembles that of dimeric EcDsbG, which does not confer this activity. However, the HP0231 catalytic motifs (CXXC and the so-called cis-Pro loop) are identical to that of monomeric EcDsbA. To understand the functioning of HP0231, we decided to study the relations between its sequence, structure and activity through an extensive analysis of various HP0231 point mutants, using in vivo and in vitro strategies. Our work shows the crucial role of the cis-Pro loop, as changing valine to threonine in this motif completely abolishes the protein function in vivo. Functioning of HP0231 is conditioned by the combination of CXXC and the cis-Pro loop, as replacing the HP0231 CXXC motif by the motif from EcDsbG or EcDsbC results in bifunctional protein, at least in E. coli. We also showed that the dimerization domain of HP0231 ensures contact with its substrates. Moreover, the activity of this oxidase is independent on the structure of the catalytic domain. Finally, we showed that HP0231 chaperone activity is independent of its redox function.
Highlights
The ability of proteins to fold into their correct three-dimensional structure is vital for cell growth and survival
H. pylori HP0231 was the first-described periplasmic dimeric oxidoreductase that has the physiological function of catalyzing disulfide formation (Roszczenko et al, 2012)
HP0231 of Helicobacter pylori was the first-described dimeric oxidoreductase involved in disulfide bond formation (Roszczenko et al, 2012)
Summary
The ability of proteins to fold into their correct three-dimensional structure is vital for cell growth and survival. In Gram-negative bacteria, disulfide bond formation takes place in the oxidative environment of the periplasm, where it is catalyzed by a set of soluble and membrane-bound Dsb (disulfide bond) proteins. This process is critical for the correct folding and structural stability of many secreted and membrane proteins. DsbC is kept in the reduced form by an integral membrane protein, DsbD, that catalyzes the transfer of electrons from the cytoplasm to the periplasm Another membrane protein, DsbB, provides disulfides to DsbA by generating them from quinone reduction (Inaba and Ito, 2008; Cho and Collet, 2013)
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