Crystallization and initial crystallographic analysis of AafA: the major adhesive subunit of the enteroaggregative Escherichia coli AAF/II pilus

Abstract

AafA is the major adhesive pilin subunit of the aggregative adherence fimbriae (AAF) from enteroaggregative Escherichia coli, which play an important role by attaching to the host cells during the initial phase of bacterial colonization and invasion. AafA has been crystallized at pH 3.4 and diffraction data have been collected to 2.1 Å resolution. Molecular replacement was unsuccessful and selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing.

Figure 1

Schematic representation of donor-strand complemented AafA. The N-terminal 12 residues consists of a His₆ tag (green) followed by residues 11–135 of mature AafA (dark blue), a DNKQ tetrapeptide linker (orange), six residues of periplasmic signal peptide (light blue) as an additional linker sequence and finally residues 1–10 of mature AafA (dark blue) at the C-terminus. The residue numbering of AafA is shown above the figure.

Figure 2

Purification of AafA. (a) Superdex 75 (GE Healthcare) gel-filtration profile of AafA. (b) SDS–PAGE of monomeric AafA after gel filtration.

Figure 3

Representative native crystals of AafA.

Figure 4

Sequence alignment of mature AafA (UniProtKB O30595), AfaE-III (UniProtKB Q57254), DraE (UniProtKB Q7BG35) and DaaE (UniProtKB P13719). The match strength is highlighted (highest to lowest: green, cyan and grey).