The noncanonical disulfide bond as the important stabilizing element of the immunoglobulin fold of the Dr fimbrial DraE subunit.

Fimbrial adhesins of pathogenic bacteria are linear protein associates responsible for binding to the specific host cell receptors. They are assembled via the chaperone/usher pathway conserved in Gram-negative bacteria. These adhesive organelles are characterized by the high resistance to dissociation and unfolding caused by temperature or chemical denaturants. The self-complemented (SC) recombinant subunits of adhesive structures make up the minimal model used to analyze stability phenomena of these organelles. The SC subunits are both highly stabilized thermodynamically and kinetically. They are characterized by a standard free energy of unfolding of 70-80 kJ/mol and a rate constant of unfolding of 10(-17) s(-1) (half-life of unfolding of 10(8) years at 25 degrees C). The DraE subunit of Dr fimbriae is characterized by a disulfide bond that joins the beginning of the A1 strand with the end of the B strand. Such localization is unique and differentiates this protein from other proteins of the Ig-like family. Sequence analysis shows that many protein subunits of adhesive structures possess cysteines that may form a potential disulfide bond homologous to that of DraE. In this paper, we investigate the influence of this noncanonical disulfide bond on the stability of DraE-sc by constructing a DraE-sc-DeltaSS mutant protein (Cys/Ala mutant). This construct unfolds thermally at a T(m) of 65.4 degrees C, more than 20 degrees C lower than that of the native DraE-sc protein, and possesses a different unfolding mechanism. The calculated standard free energy of unfolding of DraE-sc-DeltaSS is equal to 30 +/- 5 kJ/mol. This allows us to suggest that the disulfide bond is an important stabilizing feature of many fimbrial subunits.