High Resolution Studies of the Afa/Dr Adhesin DraE and Its Interaction with Chloramphenicol

Abstract

Pathogenic Escherichia coli expressing Afa/Dr adhesins are able to cause both urinary tract and diarrheal infections. The Afa/Dr adhesins confer adherence to epithelial cells via interactions with the human complement regulating protein, decay accelerating factor (DAF or CD55). Two of the Afa/Dr adhesions, AfaE-III and DraE, differ from each other by only three residues but are reported to have several different properties. One such difference is disruption of the interaction between DraE and CD55 by chloramphenicol, whereas binding of AfaE-III to CD55 is unaffected. Here we present a crystal structure of a strand-swapped trimer of wild type DraE. We also present a crystal structure of this trimer in complex with chloramphenicol, as well as NMR data supporting the binding position of chloramphenicol within the crystal. The crystal structure reveals the precise atomic basis for the sensitivity of DraE-CD55 binding to chloramphenicol and demonstrates that in contrast to other chloramphenicol-protein complexes, drug binding is mediated via recognition of the chlorine "tail" rather than via intercalation of the benzene rings into a hydrophobic pocket.

DAF- and collagen-binding properties of chimeric Dr fimbriae

“…The b-sheet and turns/unordered components accounted for about 84 % and 16 % of the amide I band area, respectively. These FTIR data are in agreement with published structures of AfaE-dsc and DraE proteins Pettigrew et al, 2004). The IR data presented showed that the insertion of the heterogenic epitopes into the A2-B loop of the DraE subunit did not perturb the Ig-barrel structure of the adhesin to any detectable extent.…”

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

“…These adhesins are assembled via the chaperone-usher pathway (260,520). The DraE adhesin subunit expresses two separate adhesion domains; the first recognizes the human decay-accelerating factor (hDAF) at the complement control protein (CCP) 2 and 3 domains (521)(522)(523)(524), and the second recognizes the N domains of several hCEACAMs (525)(526)(527)(528)(529). The physiological role of hDAF is to inhibit the complement cascade at the level of the critical C3 convertase step for the protection of normal cells from complement-mediated attack during innate activation, and in addition hDAF serves as a receptor for certain strains of pathogenic E. coli, Helicobacter pylori, and certain types of enteroviruses (530).…”

Role of Src Kinases in Mobilization of Glycosylphosphatidylinositol-Anchored Decay-Accelerating Factor by Dr Fimbria-Positive Adhering Bacteria

“…Structure-function analysis of Dr adhesin has been performed using various different mutants (11,30,32,44,45,61). One of them, the mutant strain Dr ϩ D54C (in which aspartic acid 54 is replaced by cysteine), retains hDAF-binding capability (11) but has lost the ability to mobilize hDAF around adhering Drpositive bacteria (23).…”

“…The DraE adhesin subunit has a distinct hCEACAM member binding site located primarily in its A, B, E, and D strands, located opposite the beta sheet encompassing the binding site for hDAF, indicating that the adhesin subunit can bind simultaneously to both receptors on the epithelial cell surface (31). Conformational receptor-binding studies have been conducted in order to identify the amino acid sequences in the major structural DraE and DaaE adhesin subunits engaged in receptor recognition (11,30,44,45,62).…”