Preclusion of Irreversible Destruction of Dr Adhesin Structures by a High Activation Barrier for the Unfolding Stage of the Fimbrial DraE Subunit

Abstract

Dr fimbriae of uropathogenic Eschericha coli strains are an example of surface-located adhesive structures assembled via the chaperone-usher pathway. These structures are crucial for specific attachment of bacteria to host receptors. Dr fimbriae are linear associates of DraE proteins, the structure of which is determined by a donor strand complementation between the consecutive subunits. The biogenesis of these structures is dependent on a function of the specific periplasmic chaperone and outer membrane usher proteins. In a consequence of these structural and assembly properties the potential unfolding of a single subunit in a linear associate would cause a destruction of fimbrial adhesion function. This correlates with the observed high resistance of fimbrial structures for denaturation. In this paper we show that the mechanism of thermal denaturation of DraE-sc protein is well described by an irreversible two-state model which is the reduced form of a Lumry-Eyring protein denaturation model. In theory of this model the observed stability of DraE-sc protein is determined by the high activation barrier for the unfolding stage N-->U. The microcalorimetry experiments permit to determine kinetic parameters of the DraE-sc unfolding process: energy of activation of 463.5 +/- 20.8 kJ.mol(-1) and rate constant of order 10(-17) s(-1). This corresponds to the dissociation/unfolding half-life of Dr fimbriae of 10(8) years at 25 degrees C. The FT-IR experiments show that the high stability of DraE is determined by the cooperative rigid protein core. The presented mechanism of kinetic stability of Dr fimbriae is probably universal to adhesive structures of the chaperone-usher type.

Structure, Folding and Stability of FimA, the Main Structural Subunit of Type 1 Pili from Uropathogenic Escherichia coli Strains

“…The self-complemented construct FimAa, in which antiparallel donor strand insertion is enforced (see below), shows the same nonequilibrium behavior in protein folding (unattained two-state equilibrium) that has so far been observed for all other pilus subunit constructs with artificial C-terminal donor strand. 15,20,[33][34][35] However, FimAa shows by far the highest stability and slowest folding and unfolding kinetics relative to the other self-complemented pilus domains investigated so far. 15,20,[33][34][35] NMR structure of FimAa Based on the stability data above on FimAwt and FimAt, the construct FimAa has alternative folding possibilities in that it can incorporate either its N-or C-terminal donor strand segment into its tertiary structure.…”

“…15,20,[33][34][35] However, FimAa shows by far the highest stability and slowest folding and unfolding kinetics relative to the other self-complemented pilus domains investigated so far. 15,20,[33][34][35] NMR structure of FimAa Based on the stability data above on FimAwt and FimAt, the construct FimAa has alternative folding possibilities in that it can incorporate either its N-or C-terminal donor strand segment into its tertiary structure. To confirm the anticipated incorporation of the C-terminal extension into the donor strand binding groove and to study the structural details of donor strand complementation in the type 1 pilus rod, we solved the solution structure of FimAa with NMR spectroscopy.…”

“…, showing that FimAa is a thermodynamically extremely stable protein with very high activation energy barriers for unfolding and refolding, analogous to previously characterized, selfcomplemented pilus subunits with C-terminal donor strand. 15,20,[33][34][35] The thermal unfolding transition of FimAa shows a very high melting temperature (T m ) of 87.8°C, while the moderately stable FimAwt exhibits a T m of 46.1°C (Fig. 4d).…”

“…4,[13][14][15][16][17][18][19][20]35 The artificial C-terminal extension (G-strand in the solved NMR structure) complements the Ig-fold in a canonical manner (i.e., runs antiparallel with the F-strand). The fold is mainly determined by two β-sheets, the first being composed of strand A (29)(30)(31)(32)(33)(34)(35)(36)(37)(38), the donor strand G (167-182), strand F (146-159) and strand C (67-75), and the second being composed of strands B′ (45)(46)(47)(48)(49), B″ (52-62), E (127-141), D′ (99-105) and D″ (122-124), that are packed against each other in a wound fashion (Fig. 5b and c).…”

Large Is Fast, Small Is Tight: Determinants of Speed and Affinity in Subunit Capture by a Periplasmic Chaperone

“…3a). In contrast to subunit:subunit interactions that have been suggested to have dissociation halftimes in the order of billions of years, 22,23 Caf1M-Caf1 binding is highly dynamic and is not exceptionally tight ( Table 1, mutations in the Caf1M chaperone).…”