The Noncanonical Disulfide Bond as the Important Stabilizing Element of the Immunoglobulin Fold of the Dr Fimbrial DraE Subunit

Abstract

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.

Analysis of the unique structural and physicochemical properties of the DraD/AfaD invasin in the context of its belonging to the family of chaperone/usher type fimbrial subunits

“…1) The DraD protein possesses the disulfide bond connecting the B and F strands that is unique to the family of fimbrial subunits. This renders that the DraD invasin may be used in some extent as an additional natural verification of previously presented thesis that the disulfide bond connecting A and B strands is responsible for the observed high kinetic stability of the DraE-sc [13]. 2) The network of the inter-strands main-chain hydrogen bonds stabilizing the β-barrel structure of the DraD is defective in the region of acceptor cleft.…”

“…It is rather an effect of a global optimization of the interactions that stabilize the Ig-barrel structure [4,5]. The second mechanism is based on a subunit kinetic stability denoted by a high energy of activation of the unfolding stage [12,13]. The self-complemented AfaE-sc subunit of Afa-III adhesin (98% identity to DraE subunit of Dr fimbriae) is characterized by the moderate unfolding free energy ΔG 25°C of 45.1 ± 1.5 kJ mol -1 [14].…”

“…DraE and many other fimbrial subunits are characterized by a disulfide bond, which is uniquely localized compared to other members of the Ig-type protein superfamily, as it connects the A and B strands. Our previous work showed that the DraE-sc protein with cysteine residues changed to alanine lost its kinetic stability [13]. This suggests that such a localization of the disulfide bond is important for generation of the kinetic stability of the fimbrial proteins.…”

“…In contrast to the canonical Ig-fold, position of the disulfide bridge in many fimbrial subunits is unusual since it joins two adjacent strands of the same sheet of the β-sandwich, namely the beginning of A strand with the end of B strand [13]. Beside the atypical position in the sequence also the spatial location of the bridge is unusual since it is placed at the top of the β-barrel unlike in the canonical Ig-structures, where the disulfide bond joins the strands in the middle thus positioning itself in the hydrophobic core center of the protein.…”

Structural Insight into Host Recognition by Aggregative Adherence Fimbriae of Enteroaggregative Escherichia coli

“…1) [33], [34]. As the first cysteine often marks the beginning of the subunit core structure, we hypothesized that the N-terminal sequences 12–24 residues preceding the cysteine in the major subunits could act as donor strands connecting subunits in the fiber.…”