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Mol Microbiol. Author manuscript; available in PMC 2009 January 21.
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Mol Microbiol. 2006 October; 62(2): 356–366.
Published online 2006 September 8. doi: 10.1111/j.1365-2958.2006.05375.x.
PMCID: PMC2628978
UKMSID: UKMS3383
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The solution structure of the invasive tip complex from Afa/Dr fibrils
Ernesto Cota,1 Celine Jones,2 Peter Simpson,1 Harri Altroff,2 Kirstine L. Anderson,1 Laurence du Merle,3 Julie Guignot,4 Alain Servin,4 Chantal Le Bouguénec,3 Helen Mardon,2 and Stephen Matthews1
1Division of Molecular Biosciences, Biochemistry Building, Imperial College London, South Kensington, London SW7 2AZ, UK
2Nuffield Dept. Obstetrics and Gynaecology, Division of Medical Sciences, University of Oxford Women’s Centre, Level 3, John Radcliffe Hospital, Headington, Oxford, OX3 9DU
3Unite de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris CEDEX 15, France
4Institut National de la Sante et de la Recherche Medicale, Unite 510, Faculte de Pharmacie Paris XI, Chatenay-Malabry, France
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Summary
Afa/Dr family of adhesins are produced by pathogenic E. coli strains that are especially prevalent in chronic diarrheal and recurrent urinary tract infections. Most notably, they are found in up to 50% of cystitis cases in children and 30% of pyelonephritis in pregnant women. Afa/Dr adhesins are capped surface fibrils that mediate recognition of the host and subsequent bacterial internalisation. Using the newly solved three-dimensional structure of the minimal invasive complex (AfaDE) combined with biochemical and cellular assays, we reveal the architecture of the fibrillar cap and identify a novel mode of synergistic integrin recognition.
Diffusely-adherent E. coli (DAEC) strains are present in a large proportion of patients with recurrent urinary tract infections and diarrhea. These infections often manifest as cystitis and pyelonephritis, and are especially prevalent in pregnant women and young children. Among all E. coli pathogens, DAEC serotypes can be distinguished by their diffuse pattern of adherence to epithelial Hep-2 and HeLa cells in vitro (Servin, 2005)
An important feature of DAEC strains is the presence of the Afa/Dr group of adhesins. Afa/Dr adhesins display an afimbrial/fimbrial morphology and are exported to the bacterial surface by the chaperone-usher pathway, a widespread system among gram-negative pathogens for the secretion of fimbrial proteins (Sauer et al., 2000). These adhesins are the main virulence determinants in DAEC and account for the recognition of different host cell receptors. Prominent examples are the AfaE adhesin and the AfaD invasin of the plasmid-borne afa operons (Le Bouguenec et al., 2001). AfaE mediates the primary adhesion event via binding to GPI-anchored molecules, the decay accelerating factor (DAF or CD55) (Nowicki et al., 1990) and some carcinoembryonic antigen-related molecules (CEACAMs) (Berger et al., 2004; Guignot et al., 2000). β1 integrins are also recruited by the AfaD invasin around Afa/Dr adherent bacteria, an essential interaction for the subsequent internalisation of bacteria and the maintenance of chronic infections (Guignot et al., 2001; Plancon et al., 2003).
Understanding how innate immunity responses are triggered by the interaction of DAEC with epithelial cells requires a detailed description of the fimbrial architecture and the interplay between fimbrial adhesins and host cell receptors. Our recent structural work revealed that AfaE acts as a pilin domain that assembles into a flexible fibre via the chaperone-usher pathway (Anderson et al., 2004a). An absent β-strand is initially provided by the chaperone in a process termed donor strand complementation (DSC) (Sauer et al., 2000). Subunits destined to join the lengthening fibre possess a free N-terminal strand that takes over the role previously performed by the chaperone (donor strand exchange - DSE). It has been proposed that the AfaD protein caps the AfaE fibrils, where it could efficiently perform its role as an invasin, however, the architecture of the polymer tip and its interaction with integrins is still poorly understood (Anderson et al., 2004a). To address this, we have determined the high resolution structure of an active tip complex between AfaD and AfaE, and characterised a novel synergistic interaction with α5β1 and αvβ3 integrins.
Production and functional characterization of AfaDE-dsc
We have previously described how the construction of a monomeric, self-complemented form of the AfaE-III adhesin from the afa-3 operon (AfaE-dsc) facilitated the characterisation of its structural and functional properties (Anderson et al., 2004a; Anderson et al., 2004b). In a similar manner, to investigate the optimal invasive region of the Afa/Dr adhesins, we constructed donor strand self-complemented versions of AfaD-III from the afa-3 gene cluster (Garcia et al., 1994; Le Bouguenec et al., 1993) (AfaD-dsc) and a complex between AfaD-III and AfaE-III (AfaDE-dsc). The AfaD invasin does not possess an N-terminal donor strand, which defines its role as a fimbrial capping domain (Anderson et al., 2004a; Cota et al., 2004). Construction of AfaD-dsc involved the extension of its C-terminal sequence with the N-terminal donor strand from AfaE (first 16 residues of the mature protein) via a 4-residue linker, DNKQ (Anderson et al., 2004a; Anderson et al., 2004b; Cota et al., 2004). Similarly, a construct of the AfaDE-dsc complex was created by fusion of the intact AfaD-dsc and AfaE-dsc coding sequences (Fig. 3A and 3BFigure 3).
Figure 3
Figure 3
Figure 3
A) Amino acid sequence and localisation of β-strands (black bars) in AfaDE-dsc. The mature AfaD sequence is shown in light green and mature AfaE in light blue. The N-terminal donor strand from AfaE is shown in bold, dark blue and linker regions (more ...)
We then tested the ability of these constructs to promote host-cell entry. Carboxylated polystyrene beads were covalently coupled to AfaDE-dsc, AfaD-dsc or AfaE-dsc. AfaE-dsc beads, like the bacteria producing only AfaE-III, adhered to the HeLa cells but were never observed internalized (Anderson et al., 2004a). The AfaD-dsc beads exhibited a limited interaction with cells and no significant internalization was observed (Data not shown). In contrast, beads coated with AfaDE-dsc were observed both interacting with the cell surface and internalized into the HeLa cells (Fig. 1AFigure 1). The β1 subunit of integrins has been shown to be recruited around adherent bacterial harboring afa operons (Guignot et al., 2001; Plancon et al., 2003). Immunofluorescence labeling and confocal microscopy analysis was therefore used to determine whether β1 integrin is recruited around cell-associated AfaDE-coated beads. HeLa cells incubated with AfaDE-coated beads exhibit a clear clustering of β1 integrin molecules beneath cell-associated beads, which can be seen as forming rings delineating their circumference (Fig. 1BFigure 1). Clustering of an unrelated control receptor was not observed confirming the specificity of the integrin interaction. Taken together these data (Fig. 1Figure 1) confirm that only AfaDE-dsc coated beads displayed a host cell invasion pattern similar to Afa/Dr-associated bacteria, indicating that it provides all the necessary molecular interactions for attachment and invasion.
Figure 1
Figure 1
Figure 1
A) Microscopic examination of AfaDE-coated beads with HeLa cells. Extracellular beads were labelled with rabbit anti-His6-AfaD-III and were visualized by the green fluorescence of FITC-labelled anti-rabbit IgG antibodies. Intracellular beads were visualized (more ...)
The solution structure of AfaDE-dsc
We also determined the high resolution three dimensional structures of AfaD-dsc, and AfaDE-dsc, by NMR (Figs. ​(Figs.22Figure 2-​-4).4Figure 4). Using a final combination of manual and automated assignment methods, NOESY data were comprehensively analyzed for AfaD-dsc and final family of solution structures was calculated. NMR spectra of AfaDE-dsc are very similar to of its free component subunits indicating that the domains structures are unchanged; specifically, the majority of the chemical shift differences between free AfaD/AfaE and AfaDE-dsc are limited to the domain-domain interface (Fig. 2Figure 2). Based on refined structures of AfaD-dsc and AfaE-dsc together with 150 residual dipolar coupling measurements recording on AfaDE-dsc, a high resolution structure for an AfaDE-dsc complex was finally derived, revealing a well-defined, extended domain arrangement (Figs. ​(Figs.3,3Figure 3, ​,44Figure 4 and Table 1). The final structure of the AfaD domain exhibits an Immunoglobulin-like topology (Figs ​(Figs33Figure 3 and ​and4),4Figure 4), in which the two β-sheets pack against each other in a similar fashion to the AfaE subunit. Furthermore, AfaD superimposes with an RMSD of 2.6 Å over 115 equivalent backbone Cβ atoms of the related invasin DraD. Subtle differences occur in loops D-D’ and C-C’, and in the donor strand position which is replaced by a non-native histidine tag in the DraD crystal structure (Jedrzejczak et al., 2006). An effective isotropic overall correlation time of ~16 ns for AfaDE-dsc, (~8 ns AfaD-dsc and AfaE-dsc alone) was estimated from T1 and T2​ 15N relaxation data (data not shown). Further analysis, using the program TENSOR, revealed that an axially symmetric diffusion tensor with D||/D[perpendicular]=1.95 yielded the best description of the data, confirming that in solution the average AfaDE-dsc structure approximates a cigar shape. The buried surface area at the AfaDE domain interface is 325 ± 80 Å2, which is comparable to the 330 Å2 buried by the FnIII9-10 interface of fibronectin (Leahy et al., 1996). Some interdomain flexibility at the 9/10 interface is important in maximizing the integrin interactions from the two domains and our structures suggests that integrin recognition by AfaDE may be similar in this respect.
Figure 2
Figure 2
Figure 2
An assigned 1H-15N HSQC NMR spectrum of the 32kDa AfaDE-dsc at pH 7 and 303 K (black). Spectra of AfaD-dsc (green) and AfaE-dsc (blue) are also shown for comparison. Key spectral differences are annotated and congregate at the AfaDE interface indicating (more ...)
Figure 4
Figure 4
Figure 4
A) Cα traces representing the ensemble of NMR-derived structures for AfaDE-dsc (AfaD, green; AfaE, blue).
Table 1
Table 1
Structural statistics for AfaE-dsc, AfaD-dsc and AfaDE-dsc
AfaD exhibits an Immunoglobulin-like topology, in which the two β-sheets pack against each other in an analogous fashion to AfaE and archetypal pilin domains; superimposition over equivalent residues gives RMSDs ~2.5 Å. The structure also illustrates that the free N-terminus in AfaE contributes a complementing strand to the fold of AfaD and provides high resolution detail for the incorporation of AfaD into the AfaE polymer. The conformation of the incoming strand reveals an identical strand-strand ‘register’ in both domains, highlighted by a conserved donor strand cleft that accommodates the side-chains of C-terminal Thr and Leu residues (TTKLTVT) (Fig. 5Figure 5). Interestingly, this pocket is open to solvent in AfaD but closed in AfaE, which correlates with the weaker interaction between the donor strand and AfaD, and its ability to detach itself from the bacterial fibrils in culture (Gounon et al., 2000; Jouve et al., 1997). Furthermore, AfaD does not possess a free N-terminal extension which defines its role as the initiator of fimbrial assembly and therefore the cap of the fibrillar structure.
Figure 5
Figure 5
Figure 5
A) Donor strand binding cleft in AfaD. The body of the protein is represented using a surface map (AfaD green and complementary F and A strands in white). The AfaE donor strand is shown in wire representation with side chains of residues TTKLTVT as red (more ...)
AfaDE-dsc displays synergistic binding to α5β1 and αvβ3 integrins
The integrin binding capacities of AfaD-dsc, AfaD-dsc and AfaDE-dsc were subsequently compared using a series of ELISA-based binding assays. Figure 6Figure 6 illustrates data revealing direct interactions between AfaDE-dsc and two integrin families present on both epithelial cells and enterocytes, α5β1 and αvβ3, which have also been identified as targets for other pathogens (Boyle and Finlay, 2003; Zarate et al., 2004). The interaction between AfaDE-dsc and integrin has a Kd ~ 700 nM (Table 2). This affinity is significantly lower than that measured for surface proteins from truly invasive bacteria: the invasin from Yersinia pseudotuberculosis binds the α5β1 integrin with a Kd = 5 nM (Van Nhieu and Isberg, 1991). The comparatively low affinity of AfaDE-dsc for integrins correlates with the low levels of invasion observed for Afa/Dr bacteria and their subsequent ability to persist within a host for long periods of time. The eventual release of bacteria into the lumen or urine provides a mechanism for establishing chronic/recurrent intestinal or urinary tract infections.
Figure 6
Figure 6
Figure 6
A) Binding experiments illustrating the interaction between α5β1 integrin with FnIII9-10, AfaDE-dsc, AfaD-dsc, AfaE-dsc. A fit of these data is shown yielding estimates for the dissociation constants (Table 2). No binding could be observed (more ...)
Table 2
Table 2
Calculated dissociation constants for integrin-Afa/Dr adhesins interaction
Surprisingly, a markedly weaker affinity for integrins (>10 fold higher Kd) is observed for AfaD in isolation, where as AfaE alone provides no measurable interaction (Fig. 6Figure 6). To test whether the increased integrin-binding affinity for AfaDE-dsc was a consequence of a second site interaction or enhanced AfaD domain stabilization, we performed equilibrium denaturation experiments (Fig. 7Figure 7). While the AfaD monomer is significantly less stable than AfaE, neither domain is substantially stabilized upon formation of the complex, implying that the increased binding affinity is most likely due to a weak second site interaction from AfaE and/or the AfaDE interface.
Figure 7
Figure 7
Figure 7
A) Plot of relative fluorescence intensity at different wavelengths for native and denatured forms of AfaD-dsc, AfaE-dsc AfaDE-dsc. Data were taken at the start of the transition region (at 0.286 denaturant activity units, DA, for AfaD-dsc and AfaDE- (more ...)
Cell entry by Afa-associated E. coli strains is primarily mediated by the recruitment of integrins (Guignot et al., 2001; Plancon et al., 2003). In this process, the AfaD invasin is directly involved in the recognition of the integrin β1 chain (Plançon et al., 2003). Integrins are a family of cell surface receptors responsible for interactions between the cell surfaces and the extracellular matrix as well as several important cell-cell adhesion events (Hynes, 1992). The binding properties and the arrangement of domains in the AfaDE complex are reminiscent of the synergistic, ‘two-site’ mode of integrin α5β1 binding by fibronectin and invasin from Y. pseudotuberculosis (Leong et al., 1995). Cell attachment by fibronectin occurs primarily via an RGD-loop motif from FIII10, while maximal binding and subsequent integrin activation is provided by the synergy region from the adjacent FnIII9 (Copie et al., 1998; Takagi et al., 2003). However, the exact nature of the contribution of FIII9 to maximal α5β1 activity and signaling is not clear (Altroff et al., 2004). In the invasin from Y. pseudotuberculosis two separate motifs from adjacent domains are critical for efficient integrin binding and invasion (Hamburger et al., 1999; Leong et al., 1995).
In addition to the RGD motif, integrin ligands can bind via RGD-like sequences (reviewed in (Ruoslahti, 1996)). A phage-display peptide library used to identify RGD mimetics has found variants with comparable affinity to the canonical RGD, notably those containing the DGR and NGR tripeptides (Koivunen et al., 1993). Initial inspection of the sequence alignment for the AfaD family of invasins suggested a similar binding mechanism might exist, as they contain such tripeptides in five different loop regions. In particular, the invasin used in this study, AfaD-III contains prominent DGR tripeptides in loops between strands C1-C2 and C2-D (residues 46-48 and 58-60, respectively), and an RDG sequence between residues 17-19. We have shown that non-conservative substitutions in these motifs have no significant effect on integrin binding (Manuscript in preparation). These results suggest that AfaDE binding to integrins is mediated by a non-sequential motif, such as the one previously described for the Y. pseudotuberculosis invasin (Hamburger et al., 1999; Leong et al., 1995), in which two aspartic acids separated by 100 amino acids are critical for binding. Additional mutagenesis and NMR-based experiments are in progress to define the exact nature of this interaction.
Alternative mechanisms of cell internalisation by Afa/Dr strains have also been described. Garcia et al. (2000) have shown that strains bearing the afa-3 operon can trigger cell invasion at reduced levels in the absence of the AfaD invasin. Similarly, it has been shown that binding of the Dr adhesin (97% identity with the AfaE-III adhesin) is able to promote cell invasion via the short consensus repeat 3 domain of the GPI-anchored decay accelerating factor, DAF (Selvarangan et al., 2000). The affinity of this interaction has been characterised and falls within the micromolar range (16μM) (Anderson et al., 2004a). Details regarding the specificity of each mechanism to different cell types and their relative contribution to the invasion process remain to be elucidated.
The ability to form a stable fimbriae and the large ΔGD-N of monomeric AfaE (AfaE-dsc) show that residues involved in the donor strand ‘cleft’ provide the optimal interactions for efficient polymerisation. This tight interaction is in contrast with the weaker binding of the donor strand by AfaD, as shown by the limited number of contacts and a more exposed cleft. The plasticity of this region is further highlighted by the structure of a DraD in which the cleft accommodates the terminal histidine tag of an adjacent monomer in the crystal (Jedrzejczak et al., 2006). In summary, the low levels of invasion, the relatively low binding Kd of AfaE and AfaD to receptors, the reduced stability of the AfaD monomer and its release from the bacterial fibril provide a molecular basis for the chronic and recurrent nature of infections caused by this pathogen.
Cloning, Expression and Purification of AfaE-dsc, AfaD-dsc and AfaDE-dsc
The ability to self complement AfaD and AfaE without altering their structure provides us with the opportunity of synthesising the multisubunit complexes as single polypeptide chains (Anderson et al., 2004b; Cota et al., 2004). This can be accomplished by extending the mature sequence of AfaD-III (122aa residues) (Garcia et al., 1994; Le Bouguenec et al., 1993) with a full-length, self-complemented AfaE creating the AfaDE-dsc (285aa residues, Fig. 3AFigure 3). The constructs, AfaE-dsc, AfaD-dsc and AfaDE-dsc were expressed cytoplasmically using the pRSETA plasmid (Promega) in the BL21(DE3) E. coli strain (Novagen). 15N, 13C double-labelled samples of AfaE-dsc and AfaD-dsc were produced in minimal media, containing 0.07% 15NH4Cl and 0.2% 13C-glucose, supplemented with 50 μg/ml ampicillin. A 2H, 15N, 13C triple-labelled sample of AfaDE-dsc was produced from Silantes media E. coli CDN OD1. All samples were purified in denaturing conditions (50 mM sodium phosphate buffer, pH 8.0, 0.3 M NaCl and 8 M urea) using the binding of the N-terminal hexahistidine tag to Nickel-bound agarose beads. Purified proteins were refolded by dialysis into 50 mM sodium phosphate buffer, pH 7.0 and concentrated to approximately 0.5 mM for NMR.
NMR Spectroscopy and Structure Calculation
Backbone and side-chain assignments on individual domains were completed using standard double and triple-resonance assignment methodology (Sattler et al., 1999). Hα and Hβ assignments were obtained using HBHA(CBCACO)NH (Sattler et al., 1999). The side-chain assignments were completed using HCCH-total correlation (TOCSY) spectroscopy and (H)CC(CO)NH TOCSY (Sattler et al., 1999). 3D 1H-15N/13C NOESY-HSQC (mixing time 100 ms at 500 MHz and 800 MHz) experiments provided the distance restraints used in the final structure calculation. Heteronuclear 1H-15N NOE data with minimal water saturation were acquired using the sequence described by Farrow et al. (Farrow et al., 1994).
The ARIA protocol (Linge et al., 2003) was used for completion of the NOE assignment and structure calculation for AfaD-dsc and AfaE-dsc. The frequency window tolerance for assigning NOEs was ±0.04 ppm and ±0.06 ppm for direct and indirect proton dimensions and ±0.7 ppm for nitrogen and carbon dimensions. The ARIA parameters, p, Tv, and Nv, were set to default values. The 15 lowest energy structures had no NOE violations greater than 0.5 Å and dihedral angle violations greater than 5°. These domain structures were subsequently refined against a comprehensive set of amide residual dipolar couplings (RDCs) recorded on monomeric samples. The full structural statistics are presented in Table 1. Additionally, an adapted HADDOCK approach (Dominguez, et al., 2003) was used for structure calculation of the AfaDE complex. The RDC-refined structures of AfaD and AfaE monomer were semi-rigidly docked under the influence of distance restraints describing the intermolecular NOEs between domains and 150 RDCs recorded on the AfaDE complex from two alignment media (70 and 80 from AfaD and AfaE, respectively). The best structures adjudged by the RDC energy term were selected for torsion angle dynamics and subsequent Cartesian dynamics in an explicit water solvent (see Table 1 and Fig. 4DFigure 4). 15N T1 and 15N T2, were measured using methods described elsewhere (Farrow et al., 1994). Relaxation delays of 50, 250, 400, 650, 950 and 1400 ms were employed for T1 measurements, and delays of 16.6, 33.2, 49.8, 66.4, 83.0, 99.6, 149.4 and 199.2 ms for T2. Relaxation analysis and derivation of diffusion tensors was performed according to standard techniques using the program TENSOR (Dosset et al., 2000).
Cell-invasion assay
The coating of beads with AfaE-dsc and immunofluorescence adherence assay were performed as described previously (Plancon et al., 2003). Fluorescent crimson carboxylated microspheres (Molecular Probes, Interchim, Montluçon, France) were coated with proteins. HeLa cells were used after 18 h of culture To analyse the entry of beads into cells after 3 h incubation period, cells were washed several times and fixed. Extracellular beads were then labelled by incubating unpermeabilized cells with rabbit anti-rAfaD-III antibodies (Garcia et al., 1996). The particles were washed three times and then incubated with FITC-labelled anti-rabbit IgG antibodies.
Integrin-clustering assay
Purified proteins (2 mg/ml) were coated onto 1μm polystyrene beads (Interfacial dynamics corporation, Interchim, France) according to the manufacturer’s instruction. For clustering assay, HeLa cells were grown at 37°C in 5% CO2 in Dulbecco’s modified Eagle medium (DMEM)/HamF12 containing glutamax and supplemented with 5% foetal calf serum (FCS), 1% sodium pyruvate and 1% non essential amino-acids. HeLa cells were used for clustering assay after 18 hrs of culture. Beads were added to the cells (107 beads /mL) for 1 hour. The cells were washed with phosphate buffer saline (PBS) 6 times then fixed in 3% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature then treated with 50 mM NH4Cl for 10 min.
For immunostaining, coverslips were washed twice in PBS containing 0.1% saponin, incubated overnight at 4 °C with primary antibodies (rabbit polyclonal anti-Dr adhesin (gift from B. Nowicki) and mouse anti-β1 integrin (clone 18, BD transduction laboratories) diluted in 10% horse serum, 0.1% saponin in PBS). Coverslips were then washed twice with 0.1% saponin in PBS and incubated for 1 hour with secondary antibodies (anti-rabbit TRITC and anti-mouse FITC, Jackson). Coverslips were washed twice in 0.1% saponin in PBS, once in PBS and once in H2O, and mounted in Dakocytomation fluorescent mounting medium (Dako). Samples were analysed using a confocal laser scanning microscope (LSM510, Zeiss). Images were processed using Adobe photoshop 5.0.
Integrin-binding assays
Solid phase binding assays were performed as described previously with purified placental integrins αvβ3 and α5β1 (Altroff et al., 2001; Altroff et al., 2004). Human placentas, obtained with informed consent and in accordance with the requirements of the Oxford Research Ethics Committee. Assays were performed in triplicate, and background antibody binding in the absence of ligand was subtracted from the readings. Nonspecific binding of His-tag fusion proteins to uncoated wells containing BSA only was measured separately for each ligand concentration point and subsequently subtracted from the corresponding values for total binding. Dose-response data from the assays were analyzed by non-linear regression using a sigmoidal curve fit (Prism, GraphPad Software).
Equilibrium denaturation experiments
Fluorescence measurements (excitation at 278 nm and emission at 360 nm) were performed in 50 mM sodium phosphate buffer, pH 7.0, 50 mM NaCl and a range of guanidine thiocyanate (GuSCN) concentrations at 25°C. For all proteins, final protein concentration was 3 μM. ΔGD-N values were determined with the method described by Clarke and Fersht (Clarke and Fersht, 1993). For the analysis of fluorescence data, denaturant activity units, rather than molar concentration of GuSCN, have been used, as this method yields a constant m-value at all GuSCN concentrations and therefore an accurate determination of ΔGD-N values in the absence of denaturant (Cota and Clarke, 2000; Pandya et al., 1999).
Acknowledgements
The authors are indebted for the financial support of The Wellcome Trust and the Medical Research Council. The authors would also like to thank Geoff Kelly and Tom Frenkiel of the 800MHz NMR service at NIMR. Co-ordinates for the NMR structures of AfaD and AfaDE are deposited at the Protein Databank under the accession codes 2FVN and 2IXQ.
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