Molecular Cell, Vol. 15, 647–657, August 27, 2004, Copyright 2004 by Cell Press
An Atomic Resolution Model for Assembly, Architecture, and Function of the Dr Adhesins Kirstine L. Anderson,1,2,9 Jason Billington,3,9 David Pettigrew,3 Ernesto Cota,1,2 Peter Simpson,1,2 Pietro Roversi,3 Ho An Chen,1,2 Petri Urvil,4 Laurence du Merle,5 Paul N. Barlow,6 M. Edward Medof,7 Richard A.G. Smith,8 Bogdan Nowicki,4 Chantal Le Bougue´nec,5 Susan M. Lea,3,* and Stephen Matthews1,2,* 1 Department of Biological Sciences Wolfson Laboratories Imperial College London South Kensington London SW7 2AZ United Kingdom 2 Centre for Structural Biology Imperial College London South Kensington London SW7 2AZ United Kingdom 3 Laboratory of Molecular Biophysics Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom 4 Department of Obstetrics and Gynecology and Department of Microbiology and Immunology The University of Texas Medical Branch Galveston, Texas 77555 5 Unite de Pathoge´nie Bacte´rienne des Muqueuses Institut Pasteur 28 rue du Docteur Roux 75724 Paris CEDEX 15 France 6 Edinburgh Protein Interaction Centre University of Edinburgh Edinburgh EH9 3JJ Scotland 7 Institute of Pathology Case Western Reserve University School of Medicine Cleveland, Ohio 44106 8 Adprotech Ltd. Chesterford Research Park Little Chesterford Saffron Walden Essex CB10 1XL United Kingdom
Summary Pathogenic bacteria possess adhesion protein complexes that play essential roles in targeting host cells and in propagating infection. Although each family of adhesion proteins is generally associated with a specific human disease, the Dr family from Escherichia *Correspondence:
[email protected] (S.M.L.); s.j.matthews@ imperial.ac.uk (S.M.) 9 These authors contributed equally to this work.
coli is a notable exception, as its members are associated with both diarrheal and urinary tract infections. These proteins are reported to form both fimbrial and afimbrial structures at the bacterial cell surface and target a common host cell receptor, the decay-accelerating factor (DAF or CD55). Using the newly solved three-dimensional structure of AfaE, we have constructed a robust atomic resolution model that reveals the structural basis for assembly by donor strand complementation and for the architecture of capped surface fibers. Introduction The ability of pathogenic Escherichia coli to adhere to and invade mucosal epithelial layers in humans and other mammals is an essential prerequisite for disease. Bacterial proteins—called adhesins and invasins—are involved in targeting the host. They are located on extended cell surface appendages known as fimbriae, as individual membrane-tethered molecules, or as part of an amorphous outer membrane-associated structure termed an afimbrial sheath (Soto and Hultgren, 1999). The Dr family of adhesins has been the subject of recent interest, as it was believed to contain representatives having both fimbrial and afimbrial architectures (Bilge et al., 1989; Labigne-Roussel and Falkow, 1988; LabigneRoussel et al., 1984; Nowicki et al., 1989). The Dr adhesins mediate a diffuse adherence pattern of bacteria to epithelial cells by virtue of binding to decay-accelerating factor (DAF or CD55), a 70 kDa glycophosphatidylinositol (GPI)-anchored glycoprotein that regulates complement activation (Medof et al., 1984, 1985). Members of this family are commonly isolated from uropathogenic E. coli (UPEC), which causes urinary tract infections, and diffusely adherent E. coli (DAEC), a cause of intestinal infections (Le Bouguenec et al., 2001). The afimbrial classification for some members of the Dr family was based on the fact that surface fimbriae could not be detected by electron microscopy (EM) (Garcia et al., 1996; Labigne-Roussel and Falkow, 1988; Labigne-Roussel et al., 1984; Le Bouguenec et al., 1993). Other Dr family members such as the Dr hemagglutinin and F1845 fimbriae (Bilge et al., 1989; Swanson et al., 1991), however, have been subsequently shown to exhibit a fimbrial morphology. A diffusely adherent fimbrial structure was also recently discovered in enteropathogenic E. coli (EPEC) (Keller et al., 2002). Although the Dr subunits themselves possess no sequence homology with orthodox fimbrial domains, the presence of fimbrial usher and chaperone paralogs suggests a common mechanism of assembly for Dr and prototypical fimbrial adhesins (Barnhart et al., 2000; Sauer et al., 1999, 2000b). Furthermore, recent structural analysis of the Caf1 antigen from Yersinia pestis suggests that bacterial appendages assembled via the chaperone/usher pathway are fibrillar (Zavialov et al., 2003). Key members of the Dr family are the plasmid-borne afa-3 operon, which encodes proteins that assemble
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Figure 1. Characterization and Definition of the Constructs Used and Their Binding to DAF (A) Gel filtration profile of AfaE-III. Elution positions for predicted AfaE-III multimers are indicated. WT AfaE-III and AfaE-dsc traces are shown in red (top) and blue (bottom), respectively. (B) High-field methyl region of the 1D 1H NMR spectrum for AfaE-III. WT AfaE-III and AfaE-dsc are shown as red (top) and blue (bottom) profiles, respectively. (C) Primary amino acid sequences of AfaE-dsc, AfaE-III, and DraE showing the numbering schemes chosen. Amino acid positions (identified from chemical shift mapping, mutagenesis experiments, or both) implicated in DAF binding are shown in green, orange, and purple, respectively. The natural differences between AfaE-III and DraE are highlighted in blue, and an additional point mutation introduced into DraE during cloning for large-scale production is shown in yellow. The complementing strand is shaded in gray, and the engineered turn is underlined. (D) Microscopic examination of AfaE-dsc-coated beads interacting with HeLa cells. AfaE-dsc-coated beads associated with cells are visualized by light (middle) and immunofluorescence (right) microscopy. Control experiment with BSA-coated beads visualized by light microscopy (left).
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Table 1. Structural Statistics for AfaE-dsc Construct and PDB Code for Deposited Coordinates
AfaE-dsc (1RXL)
Number of Experimental Restraints
4158
Total NOE-derived Ambiguous Unambiguous Intraresidue Sequential Medium-range (|i ⫺ j| ⱕ 4) Long-range (|i ⫺ j| ⬎ 4) TALOS (φ/)
4099 1542 2557 864 587 194 912 58
RMSD from Experimental Restraints Distance (A˚) Dihedral angle (deg.)
0.020 ⫾ 0.0025 0.52 ⫾ 0.11
RMSD from Idealized Covalent Geometry Bonds (A˚) Angles (deg.)
0.0045 ⫾ 0.00013 0.59 ⫾ 0.02
Energies (kcal mol ⫺ 1) 84.8 ⫾ 22.5 43.8 ⫾ 2.5 205 ⫾ 12.4 ⫺1259 ⫾ 20.3
ENOE Ebond Eangle Evdw Coordinate RMSD (A˚) Backbone atoms in secondary structure Heavy atoms in secondary structure Backbone atoms (all residues 1–143) Heavy atoms (all residues 1–143)
0.49 0.91 0.67 1.13
⫾ ⫾ ⫾ ⫾
0.05 0.08 0.09 0.09
Results and Discussion
Ramachandran Plota Residues Residues Residues Residues
in in in in
most favored regions (%) additionally allowed regions (%) generously allowed regions (%) disallowed regions (%)
al., 1984) and prevent subsequent amplification of the complement cascade (Medof et al., 1984, 1985). The extracellular portion of DAF includes four N-terminal complement control protein modules (CCPs), alternatively known as short consensus repeats or sushi domains (Caras et al., 1987; Medof et al., 1987). CCP modules comprise short  strands in an elongated barrel-like arrangement with N and C termini at either end of the long axis. The binding of classical pathway (CP) convertases is localized within CCP-2 and CCP-3, while regulation of the alternative pathway (AP) extends through to CCP-4 (Brodbeck et al., 1996). Further mutagenesis studies have identified CCP-3 as the CCP domain central to binding of the Dr family adhesins (Hasan et al., 2002; Le Bouguenec et al., 2001; Pham et al., 1995; Selvarangan et al., 2000). The high sequence similarity of the adhesin subunits among the Dr family raises several fundamental questions: (1) How do they assemble into surface organelles? (2) What is the architecture of resultant afimbrial and fimbrial structures? (3) What is the basis for receptor binding? Despite much recent attention, the fine structural details of Dr adhesin architecture and function have yet to be characterized. To address many of these issues, we have embarked on an atomic resolution study of the Dr family. Our combined NMR and biophysical studies of an engineered monomer of AfaE-III reveal the structural basis for self-assembly into a fine flexible fiber and its interaction with DAF.
75 19 3.3 2.7
a
Structural quality was evaluated using PROCHECK_NMR (Laskowski et al., 1996).
the afimbrial sheath, and the closely related dra operon, which encodes the fimbrial Dr hemagglutinin (Le Bouguenec et al., 1993; Nowicki et al., 1989). The afa-3 operon encodes two subunit proteins, AfaE-III and AfaDIII, that are directly linked to virulence and are colocalized at the outer membrane (Garcia et al., 1996; Gounon et al., 2000). AfaE-III mediates the primary adhesion event, while AfaD-III is necessary for efficient internalization of the bacteria. The closely related dra operon encodes the fimbrial subunits DraE and DraD, collectively known as the Dr hemagglutinin. AfaE-III and the equivalent protein from the dra operon, DraE, differ in sequence by only three residues, and both confer cell adherence by binding specifically to DAF (Garcia et al., 1996; Nowicki et al., 2001). Unlike AfaE, however, DraE binds a second receptor, the 7s domain of type IV collagen (Westerlund et al., 1989). Another intriguing distinction between AfaE-III and DraE is that the latter carries a binding site for chloramphenicol that inhibits adhesion to both DAF and type IV collagen (Carnoy and Moseley, 1997; Nowicki et al., 1988; Swanson et al., 1991; Van Loy et al., 2002). The primary function of DAF is to dissociate C3 convertases that assemble on self-cell surfaces (Medof et
Determination of the Atomic Resolution Structure for the AfaE-III Subunit Large, multimeric assemblies are difficult to study using high-resolution structural techniques. The gel filtration profile of AfaE-III in combination with NMR spectra demonstrate (Figures 1A and 1B) that the wild-type (WT) adhesin is a heterogeneous oligomer and therefore does not constitute a suitable candidate for structural analysis. High-resolution structures of archetypal fimbrial components have been successfully determined (Choudhury et al., 1999; Sauer et al., 1999, 2002; Zavialov et al., 2003), and these studies provide useful clues for experimental strategies to prevent the self-assembly of the Dr adhesins. One clue is provided by the similarity between chaperones encoded by afa-3 and dra operons and the periplasmic chaperones that assist the assembly of many fibrous bacterial organelles. These function by protecting pilus subunits while they fold within the bacterial periplasm and target them to the outer membrane usher protein for export (Barnhart et al., 2000; Sauer et al., 1999, 2000b). The subunits lack an antiparallel  strand, which is provided by the chaperone as a parallel strand, a process termed donor strand complementation (DSC) (Choudhury et al., 1999; Sauer et al., 1999, 2000a; Zavialov et al., 2003). Proteins that are destined to join the lengthening fiber possess a free N-terminal strand that allows attachment to another subunit by taking over the role previously performed by the chaperone in an antiparallel arrangement, a process termed donor strand exchange (DSE) (Choudhury et al., 1999; Sauer et al., 1999, 2002; Zavialov et al., 2003).
Molecular Cell 650
Figure 2. Three-Dimensional AfaE-dsc
Structure
of
(A) Stereoview showing C␣ traces representing the ensemble of NMR-derived structures. (B) Ribbon representation of a representative structure for AfaE-dsc. All  strands are shown in blue, with the exception of the selfcomplementing strand, which is blue/white. (C) Sequence comparison of the engineered C-terminal strand of AfaE-dsc with the N-terminal residues of members of the Dr family of adhesins. The conserved G, T, and L are highlighted. (D) The binding cleft of the self-complementing strand Gd. The body of the protein is represented using a surface map, with the exception of strand Gd in AfaE-dsc, which is shown as a ball and stick representation. (E) Plot of 2D 1H-15N steady-state heteronuclear NOE value against sequence number for AfaE-dsc.
The sequence similarity among these periplasmic chaperones suggests that DSC/DSE is employed in assembly of the Dr family adhesins. Based upon this hypothesis, we engineered a self-complemented and therefore monomeric soluble form of the adhesin. We constructed this “donor strand-complemented” AfaE-III (AfaE-dsc) by removing the first 16 N-terminal amino acids from mature AfaE-III (i.e., the donor strand) and inserting them at the C terminus after a four-residue turn, thereby enabling the donor strand to fold back and stabilize the structure (Figure 1C). This work was based on the approach used in studies of FimH whereby the sequence was extended with the N-terminal peptide of FimG, producing a mono-dispersed species, as tested by light scattering (Barnhart et al., 2000). The resulting AfaE-dsc is highly soluble and exhibits a gel filtration profile consistent with a monomeric 17 kDa protein (Figure 1A). NMR analysis shows a dramatic improvement in spectra compared to the WT form of the subunit, with the substantial decrease in line widths being the outstanding feature (Figure 1B). Moreover, the 15N relaxation data (not shown) for AfaE-dsc, which provide an estimate for the overall correlation time of 10 ns, confirm its monomeric status at mM concentrations and support our hypothesis that a DSC/DSE mechanism is involved
in polymerization. Microscopic examination of AfaE-dsccoated beads revealed a host cell adherence pattern similar to WT AfaE-III (Figure 1D), and surface plasmon resonance (SPR) experiments confirmed that AfaE-dsc binds DAF with comparable affinity to WT AfaE-III. Furthermore, SPR-based competition experiments clearly demonstrate that AfaE-dsc effectively competes for the same DAF binding site as AfaE-III (Figure 5). We can therefore conclude that AfaE-dsc displays all the functional properties of AfaE-III. Using a combination of manual and ARIA NMR assignment methods for analysis of AfaE-dsc (Linge et al., 2003), a total of 4099 nuclear Overhauser effects (NOEs) were assigned in AfaE-dsc 15N/13C-edited NOESY spectra. The structure determination was also supplemented with 58 φ and dihedral angle restraints, amounting to an average of 30 restraints per residue. For the final iteration, the 15 lowest energy structures were chosen from a total of 50 calculated based on agreement with experimental data and structural quality (Table 1). All areas of secondary structure are very well defined (Figure 2A); the average pairwise root-mean-square deviation (RMSD) for the water-refined final structures is 0.49 A˚ for the backbone atoms and 0.91 A˚ for the heavy atoms of residues within secondary structure.
Assembly and Function of the Dr Adhesins 651
Figure 3. Topology Diagrams of the Atomic Structure and Proposed Topology for All Dr Family Adhesin Polymers (A) AfaE-dsc. All  strands are shown in sky blue, with the exception of the self-complementing strand, which is sky blue/white. 310 helices are shown in dark blue, and loops are shown as black lines. (B) Topology diagram for the proposed DSC/DSE-induced AfaE-III oligomer.
The final structure of the AfaE-dsc exhibits an immunoglobulin-like topology (Figures 2B and 3A), in which the two  sheets pack against each other in a similar fashion to archetypal pilin domains (Choudhury et al., 1999; Sauer et al., 1999, 2002; Zavialov et al., 2003). Despite sequence identities of only ⵑ10%, AfaE-dsc superimposes with an RMSD of 3.2 A˚ over 105 equivalent backbone C␣ atoms of PapE from the P pilus and the capsular F1 antigen from Yersinia pestis, Caf1 (Sauer et al., 2002; Zavialov et al., 2003). The structure of AfaEdsc reveals that the remodeled C terminus performs the role of the complementing strand, thereby mimicking the intermolecular interactions observed between pilin domains and consequently confirming the DSC/DSE mechanism for the assembly of an AfaE polymer (Figures 2 and 3). This provides the first high-resolution structural evidence for the engineered self-complementation (Barnhart et al., 2000). In AfaE-dsc, the key side chains of Thr11(138) and Leu13(140) (numbering is as for the native [dsc] constructs; Figure 1B) from the complementing strand are conserved throughout the Dr family of adhesins and form the necessary interactions within the core of the AfaE fold (Figures 2C and 2D). Furthermore, low 1H-15N heteronuclear NOE values for the engineered turn (residues 123–128 in AfaE-dsc) indicate that this region is highly flexible and does not induce nonnative structure (Figure 2E). Assembly and Capping of the Dr Family Adhesins Type 1 and P pili form heteropolymeric structures composed of a rigid stalk and a thin flexible extension (fibrillium) that is connected to the adhesive tip. The adhesin domain is connected to a truncated pilin subunit, which accepts a donor strand and caps the tip of the fiber.
Earlier characterization of the Dr family adhesive organelles has demonstrated that the E protein not only functions as the adhesin, which is required for interaction with the host cell, but also as the pilin, which is responsible for construction of the organelle. Our structure of AfaE-dsc implies that the organelle is constructed via a DSC/DSE-driven polymerization of E subunits (Figure 3B). Some fimbrial structures possess an adhesin at their tip that caps the growing fiber, implying that a similar mechanism may exist for the so-called afimbrial adhesive organelle. To test this hypothesis, we investigated whether the D protein could be presented at the tip of the structure, where it can efficiently perform its role as an invasin. Protein sequence analysis reveals conservation between a region within AfaD-III and the canonical  strand F from AfaE-III, which pairs with the donated Gd strand (Figure 4A). Therefore, it is reasonable to propose that this donating Gd strand, i.e., the N terminus of mature AfaE-III, would also complement an incomplete AfaDIII domain. To test this hypothesis, we performed an NMR titration experiment with AfaD-III and a peptide corresponding to the N-terminal 18 amino acids of AfaEIII (i.e., AfaEN1–18). On addition of the AfaEN1–18 peptide, a number of resonances shift to new chemical shift positions indicative of a highly structured species with a specific complex formed between AfaD-III and AfaEN1–18. Moreover, gel filtration profiles and the reduction in line widths for AfaD-III/AfaEN1–18 resonances clearly demonstrate the loss of AfaD-III aggregation (Figure 4B). Although AfaD does not possess an obvious N-terminal extension that might perform the role of a donating strand, we performed an identical titration with the first 18 amino acids of AfaD (AfaDN1–18). Both NMR and gel
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Figure 4. Dr Family Adhesin Architecture (A) Sequence comparison of a putative AfaD-III strand with F  strand from AfaE-dsc together with the AfaE-III donating strand Gd in register. (B) Gel filtration and 1D NMR spectra (right) illustrating the removal of AfaD-III aggregates upon interaction with a peptide corresponding to the N-terminal 18 amino acids of AfaE-III (AfaEN1–18) and engineering of a self-complemented AfaD (AfaD-dsc). (C) Electron micrograph of “thin” fimbrial Dr adhesin together with schematic representations.
filtration analyses confirmed that AfaD-III structure and aggregation were unaffected by AfaDN1–18. Furthermore, we have been able to construct an AfaD that is donor strand complemented with the N terminus of AfaE (AfaDdsc), which is fully folded and monomeric (Figure 4B). In summary, our data suggest that the AfaE-III adhesins assemble into a flexible fiber that provides the link between the bacterial membrane usher (C) and the invasin at the tip (D). To investigate the dimensions of this fiber, we performed negative-stained EM on adhesins purified from Dr⫹ bacterial surfaces. Thin flexible fibers (2 nm diameter) can be readily observed that are entirely consistent with our model comprising end-to-end contact between each subunit (Figure 4C) and are reminiscent of the capsular F1 antigen from Yersinia pestis, Caf1 (Zavialov et al., 2003). In addition to the predominance of thin fibers (Figure 4C), EM analysis (data not shown) has also revealed some thicker morphologies with overall dimensions larger than the linear model suggested by our NMR studies (Bilge et al., 1989; Keller et al., 2002; Swanson et al., 1991). Thick fibers are not consistent with end-to-end contact and imply that more extensive intersubunit interactions are also possible. This would rigidify the resulting rod by either the tighter coiling of a single fiber or formation of a trimeric, coiled-coil arrangement of fibers.
Characterization and Mapping of Dr Family Adhesin-DAF (CD55) Interactions Surface plasmon resonance (SPR) experiments confirmed that AfaE-dsc and native AfaE specifically bind to a construct consisting of all four CCP domains of DAF (DAF1234) with similar affinities, irrespective of whether that construct is glycosylated at the CCP-1-CCP-2 interface or not (Figure 5). Moreover, a construct consisting of CCPs 1 and 2 (DAF12) did not show detectable binding to Dr family adhesins, while constructs containing CCPs 2, 3, and 4 (DAF234), CCPs 2 and 3 (DAF23), or CCPs 3 and 4 (DAF34) bound to native AfaE with comparable affinities to the DAF1234 construct (Kd is ⵑ10 M in all cases). This observation reinforces the importance of CCP-3 for the bacterial DAF interaction. High-resolution detail for any DAF-ligand interaction has yet to be reported. The availability of 3D structures for DAF and, in this study, AfaE provides the opportunity to perform NMR titration experiments as a means to investigate the interacting surfaces (Lukacik et al., 2004; Uhrinova et al., 2003; Williams et al., 2003). An analysis of amide line width and chemical shift changes for AfaEdsc in the presence of either DAF23 or DAF1234 was carried out (Figure 6). A number of clustered amide resonances move or broaden, signifying a likely contact with the ligand, while the majority of the spectrum remains un-
Assembly and Function of the Dr Adhesins 653
Figure 5. The Interaction between DAF and AfaE (A) Binding experiments illustrating the interaction between DAF and AfaE/AfaE-dsc. SPR binding curves of the interaction between DAF1234 and AfaE. The inset shows a Scatchard plot of equilibrium responses derived from the data presented in the panel. A linear fit of these data is shown and yields an estimate for the dissociation constant (Kd) of 16 M. (B) AfaE-dsc competition for DAF binding. DAF (12.5 M) binding to AfaE-III in the absence (⫺) and presence (⫹) of AfaE-dsc (50 M). The data presented are the results of three independent experiments over two independent AfaE-III surfaces with ⵑ5800 RU (left) and ⵑ5300 RU (right) coupled on each.
changed. Severe resonance broadening that is specific to the affected amides suggests an exchange rate between free and bound states on an NMR intermediate timescale, which is entirely consistent with the measured dissociation constant (Kd is ⵑ10 M). The DAF binding region of AfaE-dsc may be delineated and lies on one side of the molecule, forming a large convex surface comprising side chains from strands A1, A2, B, C2, E, F, and Gd (Figure 6C). In agreement with the NMR data, mutations (Van Loy et al., 2002) introduced at positions D61(45), I73(57), and N77(61) (where residue numbers are shown as in the native [dsc] structures) within strands C1 and C2 have the largest effects on activity, completely abolishing DAF binding. In addition, our data are consistent with the discovery that chloramphenicol abolishes DAF binding by DraE. AfaE-III and DraE differ at only three amino acid positions [N52(36)D, M88(72)T, and T111(95)I], with the single change T111(95)I leading to the chloramphenicol-sensitive phenotype. It has therefore been postulated that this substitution (Carnoy and Moseley, 1997) permits the creation of a chloramphenicol binding pocket within the DAF binding site of the adhesin. The location of position 111(95) on the E strand within the DAF binding region corrobo-
rates this hypothesis. Its substitution with isoleucine presumably enables a productive interaction with chloramphenicol, thereby inhibiting the DAF interaction. Having revealed the surface of the adhesin that interacts with DAF, we next attempted to delineate the residues on DAF23 that form the complementary binding surface (Figure 6B). Upon mixing with AfaE, significant chemical shift perturbations are observed in both CCP domains and at the CCP-2/3 interface (Figure 6D). These data support the role of CCP-3 in AfaE binding, as many perturbed amides, notably the contiguous patch between 168 and 178 in CCP-3, are proximal to the region of a rare polymorphism in DAF, in which substitution of Ser165 with a leucine influences adhesion binding (Hasan et al., 2002). Interestingly, F169 within this patch has been shown to be particularly important in the inhibition of alternative pathway activation (Williams et al., 2003). On the other hand, chemical shift changes in CCP-2 are widely dispersed and lie on both sides of the molecule, implying either multiple interaction sites for AfaE or significant indirect binding effects. In view of the fact that AfaE binds to DAF34 but does not bind DAF12, CCP-3 contributes most to the free energy of binding. Global conformational changes such as new
Molecular Cell 654
Figure 6. Identification of the Mutual Interaction Surfaces for AfaE-dsc and DAF23 (A) A region of 2D 1H-15N HNCO spectra for 15N,13C AfaE-dsc (black) and in the presence of 15N-labeled DAF23 at the molar ratio 1:1 (red). Resonances with perturbed line widths or resonance positions are indicated. (B) Region of 2D 1H-15N HSQC spectra for 15N-labeled DAF23 (black) in presence of unlabeled AfaE-dsc at the molar ratio 1:1 (red). Resonances with perturbed line widths or resonance positions are indicated. (C) Solvent accessible surface representations of AfaE-dsc with perturbed residues colored in green. Selective assignments are labeled in order to delineate the binding surface. (D) Solvent accessible surface representation of DAF1234 (PDB code 1OJV) with perturbed residues colored in orange and assigned. Selective assignments are labeled. (E) Alternative pathway C3 convertase hemolytic assay. Data represent an average of three experiments (Z ⫽ 1.9; DAF234 ⫽ 50 ng/ml). Levels of inhibition in the presence of DAF together with increasing concentrations of AfaE-dsc are shown in blue, while data for AfaE-dsc alone are in red.
stabilizing contacts at the DAF23 interface may contribute to the diffuse chemical shift changes observed within CCP-2. Our results demonstrate that the binding regions for AfaE and the complement pathway convertases lie in close proximity to each other on DAF, which raises the
possibility that binding of Dr adhesins might interfere with complement regulatory function. In this regard, hemolytic studies with the purified AfaE-dsc adhesin showed that while its binding does not affect DAF’s activity against the classical pathway convertase C4b2a, it antagonizes DAF regulation of the alternative
Assembly and Function of the Dr Adhesins 655
pathway convertase C3bBb (Figure 6E). Thus, in addition to utilizing DAF as a convenient cellular attachment point, it is possible that Dr adhesins block or exploit some of the cellular functions of DAF—for example, making host cells more susceptible to complementmediated lysis may facilitate release and enhanced spreading of bacteria. Concluding Remarks The solution structure of AfaE-dsc, in which a self-complementing  strand was engineered into the C terminus, is strongly indicative of an analogous mode of assembly to that of fimbrial appendages. The E subunits assemble end to end to form thin filaments that are capped with the invasin (D) at the tip. The two-dimensional array implied by the “afimbrial sheath” terminology for AfaE presumably results from a collapse of the fine fibrillar structures onto the bacterial surface. The structure of AfaE, combined with binding studies and previously solved structures of DAF, has allowed the mutual interaction surfaces of the E subunit/DAF complex to be defined. Experimental Procedures Cloning, Expression, and Purification of AfaE-dsc and Native AfaE-III The construct AfaE-dsc was expressed cytoplasmically using the pRSETA plasmid (Promega) in the BL21(DE3) E. coli strain (Novagen). 15N,13C double-labeled samples of AfaE-dsc were produced in minimal media, containing 0.07% 15NH4Cl and 0.2% 13C-glucose, supplemented with 50 g/ml ampicillin. AfaE-dsc was purified in denaturing conditions (50 mM sodium phosphate buffer [pH 8.0], 0.3 M NaCl, and 8 M guanidine hydrochloride) using the binding of the N-terminal hexahistidine tag to nickel bound agarose beads. Purified protein was refolded by dialysis into 50 mM sodium acetate buffer (pH 5.0) and concentrated to approximately 1 mM for NMR. Native AfaE-III was expressed as N-terminal hexahistidine fusions in E. coli strain M15 (Qiagen). The adhesins were purified from the supernatant using nickel affinity chromatography, followed by size exclusion chromatography in an S75 Sepharose column (Pharmacia Biotech). Expression and Purification of DAF Constructs DAF constructs were overexpressed either in Pichia pastoris (DAF1234, DAF234, DAF12, DAF34 [Powell et al., 1997] and 15N-DAF23 [Uhrinova et al., 2003]) or refolded from E. coli (DAF1234 [White et al., 2004]). For the chemical shift perturbation experiments, 15N-DAF23 samples were prepared in 50 mM sodium phosphate buffer (pH 7.0) and concentrated to ⵑ0.5 mM. Surface Plasmon Resonance Native AfaE-III was covalently coupled to the carboxylated dextran matrix on the surface on an activated CM5 sensor chip using the primary amine coupling kit (BIAcore AB). After activation according to the standard protocol, 0.15 mg/ml of AfaE-III in 10 mM sodium acetate (pH 4.5) was injected. Differing levels were immobilized (5,000–15,000 RU) by varying the volume of protein injected. All interaction sensorgrams were collected at 20⬚C by flowing 80 l of the various DAF constructs at a flow rate of 20 l/min. For the competition experiments, AfaE-dsc remained in solution and competed for DAF binding to AfaE-III bound on the chip surface. The data presented are the results of three independent experiments over two AfaE-III surfaces (5800 RU and 5300 RU coupled on each surface). Each experiment was performed as matched triplets where three sequential injections were made of (1) 20 l of AfaE-dsc at a concentration of 50 M, (2) 20 l of a mixture of DAF at a concentration of 12.5 M and AfaE-dsc at 50 M, and (3) 20 l of DAF at a concentration of 12.5 M. Binding of DAF alone is shown as 100%, and the binding obtained in the presence of AfaE-dsc is given as
a percentage. These data clearly show that AfaE-dsc effectively competes for DAF binding with both AfaE-III and DraE. NMR Spectroscopy and Structure Calculation Backbone and side chain assignments were completed using standard double- and triple-resonance assignment methodology (Sattler et al., 1999). H␣ and H assignments were obtained using HBHA(CBC ACO)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 NOESYHSQC (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. (1994). A total of 321 long-range NOEs, providing unambiguous 3D information, were manually assigned from the NOESY data. The ARIA protocol (Linge et al., 2003) was used for completion of the NOE assignment and structure calculation. A total of 4099 NOE-derived distances were assigned from 13C- and 15N-edited spectra, which comprised 2557 unambiguous and 1542 ambiguous restraints. Dihedral angle restraints derived from TALOS were also implemented. 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 A˚ and no dihedral angle violations greater than 5⬚. The structural statistics are presented in Table 1. Chemical Shift Mapping for the AfaE and DAF Interaction For NMR mapping experiments, 15N,13C-labeled AfaE-dsc was prepared in 50 mM sodium acetate buffer at pH 5.3 at approximately 30 M in 0.5 ml as mentioned above. 15N DAF in the same buffer was introduced in several steps up to a molar ratio of 1:1, and 2D 15 N-1H HNCO spectra were recorded at each stage under identical experimental conditions. The titration was repeated with 15N-labeled DAF and unlabeled AfaE-dsc, using the 2D 15N-1H HSQC experiment to detect the DAF amides. A further chemical shift mapping experiment was carried out as above using DAF1234 instead of DAF23. Cell Adherence Assay The coating of beads with AfaE-dsc and immunofluorescence adherence assay were performed as described previously (Plancon et al., 2003). Preparation and Analysis of Purified Dr Fimbrae To purify the natural, polymeric forms of the Dr adhesin, E. coli DH5 ␣ (pCC90) (Carnoy and Moseley, 1997) was spread on dried surfaces of 10 LB agar plates (diameter 150 mm) with 100 g ampicillin per ml and grown o/n at 37 C⬚. The bacteria were scraped from the plates into 15 ml of phosphate buffered saline (PBS), washed with PBS, resuspended in 15 ml of PBS, and heated at 65 C⬚ for 20 min. After 15 min of centrifugation at 18,000 g, the supernatant was precipitated by adding an equal volume of 40% (w/v) ammonium sulfate, gently mixed o/n at 4 C⬚, and pelleted at 18,000 g for 5 min. The pellet was resuspended and reprecipitated in 40% ammonium sulfate and finally resuspended into 5 ml of 0.5% of sodium deoxycholate in 4 M urea, gently mixed o/n at 4 C⬚, and purified in Sepharose 4B column (Amersham Biosciences Corp., Piscataway, NJ) with the same buffer. The Dr adhesin preparation showed a hemagglutination that was reversible by adding chloramphenicol at 10 M. For electron microscopy, five microliter drops of the dispersed purified adhesin were absorbed to carbon-coated 200 mesh Ni grids. The grids were negatively stained with 2% phosphotungstic acid (pH 6.8) for 10 s, wick dried with filter paper, and observed in a Philips 201 transmission electron microscope at 60 kV. Alternative and Classical Pathway Activation Assays Sheep erythrocytes (ESh; 1 ⫻ 109 ml⫺1) were sensitized with rabbit anti-sheep hemolysin (A) at 37⬚C for 30 min, followed by 4⬚C for 30 min. The resulting EShA (adjusted to 1 ⫻ 109 ml⫺1 in DGVB2⫹) were incubated at 30⬚C for 15 min with an equal volume of DGVB2⫹ containing 100 site-forming units (SFU) of C1. After washing, the re-
Molecular Cell 656
sulting EShAC1 (1 ⫻ 108 ml⫺1 in DGVB2⫹) were incubated at 30⬚C for 20 min with an equal volume of DGVB2⫹ containing 100 SFU of C4 to yield EShAC14. For studies of classical pathway activation, 100 l of EShAC14 (1 ⫻ 108 ml⫺1 in DGVB2⫹) were incubated at 30⬚C for 5 min with 100 l of C2. Hemolytic sites then were developed by addition of 1.3 ml of GVB-E containing 150 l guinea pig serum (C-EDTA) as a source of C3-9 and incubation at 37⬚C for 60 min. For studies of alternative pathway activation, EShAC142 prepared as above (200 l at 5 ⫻ 107 ml⫺1 in DGVB2⫹) were incubated at 30⬚C for 20 min with 100 l of DGVB2⫹ containing 2 SFU of C3. After washing, the resulting EShAC1423 (1 ⫻ 108 ml⫺1 in GVB-E) were incubated for 2–4 hr at 30⬚C until decay of C2 and C1 was complete (as assessed by the absence of lysis upon addition of C2 and C-EDTA). The resulting E⬘shAC43 (1 ⫻ 108 ml⫺1 in DGVB2⫹) then were incubated at 30⬚C for 30 min with an equal volume of DGVB2⫹ containing 1.5 SFU of factor B and factor D. Hemolytic activity was developed by addition of 1.3 ml of C-EDTA and incubation at 37⬚C for 1 hr. In all hemolytic assays, cells were pelleted and hemoglobin color was read at 412 nm.
Garcia, M.I., Gounon, P., Courcoux, P., Labigne, A., and Le Bouguenec, C. (1996). The afimbrial adhesive sheath encoded by the afa-3 gene cluster of pathogenic Escherichia coli is composed of two adhesins. Mol. Microbiol. 19, 683–693. Gounon, P., Jouve, M., and Le Bouguenec, C. (2000). Immunocytochemistry of the AfaE adhesin and AfaD invasin produced by pathogenic Escherichia coli strains during interaction of the bacteria with HeLa cells by high-resolution scanning electron microscopy. Microbes Infect. 2, 359–365. Hasan, R.J., Pawelczyk, E., Urvil, P.T., Venkatarajan, M.S., Goluszko, P., Kur, J., Selvarangan, R., Nowicki, S., Braun, W.A., and Nowicki, B.J. (2002). Structure-function analysis of decay-accelerating factor: Identification of residues important for binding of the Escherichia coli Dr adhesin and complement regulation. Infect. Immun. 70, 4485– 4493. Keller, R., Ordonez, J.G., de Oliveira, R.R., Trabulsi, L.R., Baldwin, T.J., and Knutton, S. (2002). Afa, a diffuse adherence fibrillar adhesin associated with enteropathogenic Escherichia coli. Infect. Immun. 70, 2681–2689.
Coordinates and Figure Preparation Coordinates for the ensemble of NMR structures have been deposited at the Protein Databank under the accession code 1RXL. Tables of NMR assignments and restraints are available as supplementary material and have been deposited in the BioMagResBank in Madison, WI (accession code 5947). Figures were prepared using programs AESOP (Martin Noble, unpublished program), Jplot (Jeremy Craven, unpublished program), and LIGPLOT (Wallace et al., 1995).
Labigne-Roussel, A., and Falkow, S. (1988). Distribution and degree of heterogeneity of the afimbrial-adhesin-encoding operon (afa) among uropathogenic Escherichia coli isolates. Infect. Immun. 56, 640–648.
Acknowledgments
Laskowski, R.A., Rullmann, J.A.C., MacArthur, M.W., Kaptein, R., and Thornton, J.M. (1996). AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486.
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