Journal of Biomolecular NMR 29: 411–412, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 411 Letter to the Editor: Complete resonance assignments of the ‘donor-strand complemented’ AfaD: The afimbrial invasin from Diffusely Adherent E. coli Ernesto Cota a,b , Ho An Chen a,b , Kirstine L. Anderson a,b , Peter Simpson a,b , Laurence du Merle c , Christine Bernier-F´ ebreau c , Rafal Pi¸ aatek d , Beata Zalewska d , Bogdan Nowicki e , J´ ozef Kur d , Chantal Le Bougu´ enec c & Stephen Matthews a,b,∗ a Department of Biological Sciences, Wolfson Laboratories and b Centre for Structural Biology, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AZ, U.K.; c Unite de Pathogenie Bacterienne des Muqueuses, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris CEDEX 15, France; d Department of Microbiology, Gda´ nsk University of Technology, ul. Narutowicza 11/12, 80-952 Gda´ nsk, Poland; e Department of Obstetrics & Gynaecology and Department of Microbiology and Immunology The University of Texas Medical Branch, Galveston, TX 77555-1062, U.S.A. Received 24 September 2003; Accepted 28 November 2003 Key words: AfaD, Afimbrial sheath, DAEC, DraD, integrin, NMR, UPEC Biological context Members of the Afa (Af imbrial A dhesin) family of adhesins have been isolated from Diffusely Adherent E. coli (DAEC) strains that are known to cause in- testinal disease in humans and animals. Furthermore, a diffusely adherent adhesin has also been identified in enterohaemorrhagic and enteropathogenic E. coli (Keller et al., 2002). The afimbrial sheath is composed of two proteins from the afa operon, AfaE and AfaD, that have different roles in host cell interactions. AfaE and AfaD are directly linked to virulence, co-localise at the outer membrane and perform the roles of ad- hesin and invasin, respectively (Jouve et al., 1997). The focus of this work is the AfaD-III subtype from human E. coli isolates (Le Bouguénec et al., 1993) and is identical to the DraD invasin from uropathogenic E. coli (Zalewska et al., 2001). AfaD-III was found to be dispensable in the production of functional adhes- ins but no invasion of mucosal layers could be detected (Garcia et al., 1996). The role of AfaD-III as an invasin was further confirmed by the translocation of AfaD- coated gold particles into mucosal cells (Gounon et al., 2000; Jouve et al., 1997) via an interaction with the α5β1 integrin (Guignot et al., 2001). The presence of a chaperone and usher within the afa operon suggests that AfaD and AfaE adopt donor strand complementa- ∗ To whom correspondence should be addressed. E-mail: s.j.matthews@imperial.ac.uk. tion for construction of the afimbrial sheath (Zavialov et al., 2003). Methods In order to alleviate the inherent self-association prop- erties of AfaD and facilitate isolation of soluble form a donor-strand complemented AfaD (AfaD-dsc) was constructed, based on the highly successful ap- proach for FimH (Barnhart et al., 2000). A four- residue linker was added to the C-terminus of the wild-type AfaD-III sequence followed by the 16- residue N-terminal donating peptide from AfaE-III: VVPQE-DNKQ-GFTPSGTTGTTKLTVT. This con- struct was expressed using the pQE-30 plasmid in the M15/pREP4 E. coli strain (QIAGEN). 15 N, 13 C double-labelled samples of AfaD-dsc were produced in minimal media, containing 0.07% 15 NH 4 Cl and 0.2% 13 C-glucose, supplemented with 50 μg ml −1 ampicillin. Protein expression was induced by the ad- dition of 50 μM isopropyl-β-D-thiogalactopyranoside. AfaD-dsc was purified in denaturing conditions (50mM sodium phosphate, pH 8.0, 0.3 M NaCl and 8 M urea) using the binding of the N-terminal hexahis- tidine tag (MRGSHHHHHHGS) to the Co 2+ -agarose resin Talon (CLONTECH). Purified protein was refol- ded by dialysis into 50mM sodium phosphate buffer pH 7.0 and concentrated to approximately 0.5mM for NMR.