Role of the disulfide bond in stabilizing and folding of the fimbrial protein DraE from uropathogenic Escherichia coli

Abstract

Dr fimbriae are homopolymeric adhesive organelles of uropathogenic Escherichia coli composed of DraE subunits, responsible for the attachment to host cells. These structures are characterized by enormously high stability resulting from the structural properties of an Ig-like fold of DraE. One feature of DraE and other fimbrial subunits that makes them peculiar among Ig-like domain-containing proteins is a conserved disulfide bond that joins their A and B strands. Here, we investigated how this disulfide bond affects the stability and folding/unfolding pathway of DraE. We found that the disulfide bond stabilizes self-complemented DraE (DraE-sc) by ∼50 kJ mol−1 in an exclusively thermodynamic manner, i.e. by lowering the free energy of the native state and with almost no effect on the free energy of the transition state. This finding was confirmed by experimentally determined folding and unfolding rate constants of DraE-sc and a disulfide bond-lacking DraE-sc variant. Although the folding of both proteins exhibited similar kinetics, the unfolding rate constant changed upon deletion of the disulfide bond by 10 orders of magnitude, from ∼10−17 s−1 to 10−7 s−1. Molecular simulations revealed that unfolding of the disulfide bond-lacking variant is initiated by strands A or G and that disulfide bond-mediated joining of strand A to the core strand B cooperatively stabilizes the whole protein. We also show that the disulfide bond in DraE is recognized by the DraB chaperone, indicating a mechanism that precludes the incorporation of less stable, non-oxidized DraE forms into the fimbriae.

A shear stress micromodel of urinary tract infection by the Escherichia coli producing Dr adhesin

“…87˚C, with a free energy of unfolding of 83 kJ mol -1 and a unfolding half-life of 10 8 years at 25˚C [45,46]. We concluded that this high stability, typical for adhesive structures of the chaperone-usher type, is crucial for the maintenance of integrity and functionality of Dr fimbriae [47]. However, colonization of the urinary tract by Dr+ E. coli is equally dependent on the formation of productive bonds between Dr fimbriae and host receptors.…”

Experimental approach to study the effect of mutations on the protein folding pathway

“…There are a great number of theoretical works where the protein folding pathway has been appraised and analyzed, for example, [1]. It was shown that substitutions of amino acid residues, truncation/elongation of the loops and structure elements can affect not only the protein stability but also change completely its folding pathway, i.e., the sequence of formation of different protein regions, for example, [2–6]. Unfortunately, only few experimental methods and techniques are at hand that allow studying the protein folding pathway.…”

Salmonella enterica BcfH Is a Trimeric Thioredoxin-Like Bifunctional Enzyme with Both Thiol Oxidase and Disulfide Isomerase Activities

“…Cysteine residues are present in all the fimbrial subunits of the bcf operon. The role of thiol oxidases in the folding of different fimbrial components is well documented (8,32,51). Moreover, Salmonella has a dedicated thiol oxidase SrgA essential for the biogenesis of Pef fimbriae (5).…”