Crystal structure and analysis of HdaB: The enteroaggregative Escherichia coli AAF/IV pilus tip protein

Abstract

Enteroaggregative Escherichia coli is the primary cause of pediatric diarrhea in developing countries. They utilize aggregative adherence fimbriae (AAFs) to promote initial adherence to the host intestinal mucosa, promote the formation of biofilms, and mediate host invasion. Five AAFs have been identified to date and AAF/IV is amongst the most prevalent found in clinical isolates. Here we present the X-ray crystal structure of the AAF/IV tip protein HdaB at 2.0 Å resolution. It shares high structural homology with members of the Afa/Dr superfamily of fimbriae, which are involved in host invasion. We highlight surface exposed residues that share sequence homology and propose that these may function in invasion and also non-conserved regions that could mediate HdaB specific adhesive functions.

Keywords: AAF/IV; Escherichia coli; HdaB; adhesion; chaperone-usher; fimbria; invasion; pilus.

Figure 1

The HdaB‐dsA domain‐swapped dimer. A: Gel filtration profile of monomeric (17.5 kDa) and dimeric (35 kDa) HdaB‐dsA. B: Asymmetric unit of HdaB‐dsA crystals. Upper panel: domain‐swapped dimer of HdaB‐dsA shown as cartoon with citrate ions shown as spheres. The linker region is boxed and expanded below. Lower panel: the linker regions shown as sticks and also the Cys131‐Cys131 inter‐domain disulfide bond are highlighted.

Figure 2

Overall Structure of HdaB‐dsA. A: Stereo cartoon representation of an individual HdaB‐dsA monomer with secondary structure labeled (β‐strands and loops). HdaB from chain A is colored teal whilst the HdaA donor strand from chain B is colored red. N/C‐termini are annotated as residue type/number in red (HdaA) and black (HdaB). For clarity the artificial linker is not shown. B: Topology of HdaB‐dsA colored and labeled as in (A). C: Surface representation of HdaB‐dsA with self‐complementing donor strand from HdaA as sticks. Residues for interacting side‐chains in the HdaA strand are indicated.

Figure 3

Putative functional binding regions of HdaB. A: Overlay of HdaB‐dsA from chains A and B. Regions that display significant structural variation are annotated. B: Cartoon representation of HdaB‐dsA (teal) superposed with DraD (pdb: 2axw in purple), AggB (pdb: 4phx in yellow), AafB (pdb: 2orl in green) and SefD (pdb: 3uiz in blue). Regions that display significant structural variation are annotated. C: Primary sequence alignment of HdaB (UniProtKB: B3V224), AafB (UniProtKB: D3H575), AggB (UniProtKB: P46006), Agg3B (UniProtKB: C9K5V1), DraD/AfaD (UniProtKB: Q47038) and SefD (UniProtKB: Q53997). Identical and similar amino acid residues are shaded in red and orange, respectively. Secondary structure of HdaB is shown above as lines (loops) and arrows (β‐strands), and * represents conserved residues that are exposed on the surface of HdaB. D: Upper panel: surface representation of monomeric HdaB‐dsA colored based on (C). Lower panel: electrostatic surface potential of HdaB‐dsA. Three regions with sequence conservation based on (C) are circled and labeled 1 to 3. Secondary structure within these regions are annotated as in Figure 2(B). The citrate 1/1′ binding site in HdaB_dsA is represented as a yellow star and the potential galactose binding site is shown as a black star. E: Potential binding site of galactose in HdaB. PsaA/galactose complex (pdb: 4f8p) is superimposed onto HdaB‐dsA and key residues are shown as sticks. F: Citrate 1 binding site on HdaB_dsA chain A with key residues are shown as sticks.