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High resolution studies of the Afa/Dr adhesin DraE and its interaction with chloramphenicol David Pettigrew#1, Kirstine L. Anderson#2,3 Jason Billington1, Ernesto Cota2,3, Peter Simpson2,3, Petri Urvil4, Filip Rabuzin2,3, Pietro Roversi1, Bogdan Nowicki4 Laurence du Merle5, Chantal Le Bouguénec5, Stephen Matthews2,3 and Susan M. Lea1 1Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South

Parks Road, Oxford OX1 3QU 2Department of Biological Sciences, Wolfson Laboratories, Imperial College London, South

Kensington, London SW7 2AZ, UK 3Centre for Structural Biology, Imperial College London, South Kensington, London SW7 2AZ,

UK

4Department of Obstetrics & Gynaecology and Department of Microbiology and Immunology The University of Texas Medical Branch, Galveston, TX 77555-1062, USA.

5Unite de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris CEDEX 15, France #These authors contributed equally to the work

JBC Papers in Press. Published on August 24, 2004 as Manuscript M409284200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Pathogenic E. coli expressing Afa/Dr adhesins are able to cause both urinary tract and

diarrhoeal infections. The Afa/Dr adhesins confer adherence to epithelial cells via

interactions with the human complement regulating protein, Decay Accelerating Factor

(DAF or CD55). Two of the Afa/Dr adhesions, AfaE-III and DraE, differ from each other

by only three residues but are reported to have several different properties. One such

difference is disruption of the interaction between DraE and CD55 by chloramphenicol,

whereas binding of AfaE-III to CD55 is unaffected. Here we present a crystal structure of a

strand-swapped trimer of wild type DraE. We also present a crystal structure of this trimer

in complex with chloramphenicol, as well as NMR data supporting the binding position of

chloramphenicol within the crystal. The crystal structure reveals the precise atomic basis

for the sensitivity of DraE-CD55 binding to chloramphenicol and demonstrates that, in

contrast to other chloramphenicol-protein complexes; drug-binding is mediated via

recognition of the chlorine “tail” rather than via intercalation of the benzene rings into a

hydrophobic pocket.

Diffusely Adherent Escherichia coli (DAEC) has recently been accepted as a

diarrhoeagenic strain of E. coli which causes watery diarrhoea in young children (1-3). DAEC

share a characteristic pattern of infection of epithelial cells, in which the bacteria form a diffuse

pattern around the host cells, and is accompanied by an elongation of the microvilli (4). DAEC is

also characterised by the presence of an afa operon (5) which expresses the Afa/Dr adhesion

complex on the surface of the bacteria.

Although originally thought to be afimbrial, the Afa/Dr adhesins have been shown to form

extended fimbrial structures on the surface of bacteria (5-9). These structures are made up of the

E protein, which acts as both the adhesin and the major structural subunit (10,11) and the D

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protein which caps the structure and acts as the invasin (12). They mediate location of bacteria to

the gut or urinary tract via adhesion to the mammalian complement regulatory protein, the Decay

Accelerating Factor (DAF or CD55). DAF is 70kDa protein found on the apical surface of host

epithelial cells and attaches to the cell surface via a glycophosphotidylinositol (GPI) anchor (13-

15). The Afa/Dr adhesins have also been shown to bind to members of the carcinoembryonic

antigen family, although the significance of this is yet to be determined (16,17).

The Afa/Dr adhesins comprise AfaE-I to AfaE-VIII, F1845, DraE and entropathogenic E.

coli Afa (9). Although these adhesins differ in sequence, they all bind to the third CCP domain of

DAF with the exception of AfaE-VII and AfaE-VIII (18). Two of the Afa/Dr adhesins, AfaE-III

and DraE, differ in sequence by only three amino acids but possess significantly different

properties. For example, DraE has been reported to bind to the 7s domain of type IV collagen

(19) but AfaE-III does not. The binding of DraE to both DAF and type IV collagen is blocked by

the presence of chloramphenicol (20-22) whereas the binding of AfaE-III to DAF is unaffected.

Recent structures (11) for an engineered form of the AfaE-III adhesin (AfaE-dsc) have

allowed an atomic understanding of its assembly into fimbrae and has also allowed mapping of

the sites of CD55 interaction onto the adhesin. Dr family fimbrial assembly is seen to proceed in

the same way as bacterial pili assembly (11). Briefly, adhesin subunits are found to consist of an

immunoglobulin-like fold which misses a central antiparallel β-strand and also possess an N-

terminal extension. In the bacterial periplasm the missing strand is provided by a periplasmic

chaperone in a process termed donor strand complementation (DSC) which aids folding and also

targets the adhesin subunits to the outer membrane usher protein for export (23-28). Assembly

into fimbriae on the bacterial surface proceeds via the free N-terminal strand which allows

attachment to another adhesin subunit by taking over the role previously performed by the

chaperone in an anti-parallel arrangement, a process termed donor strand exchange (DSE) (24,27-

29). Chemical shift mapping was used to map CD55 binding and localised this to one face of the

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structure (11). The engineered AfaE-dsc used for these structural studies (11,30) is maintained in

a monomeric state by relocation of the N-terminal extension to the C-terminus, where it is able to

fold back and provide the missing strand within the Ig fold—a process termed self-

complementation.

This paper presents X-ray structures for native DraE and AfaE-III in isolation and also of

a DraE-chloramphenicol complex and seeks to understand the basis for the differential receptor

and inhibitor binding exhibited by the two proteins. These structures reveal a novel mode of

chloramphenicol binding that provides potential for the design of a new class of anti-bacterial

agents.

EXPERIMENTAL PROCEDURES

Cloning, Expression and Purification of AfaE-dsc, native DraE, native AfaE-III and

CD55—Native AfaE-III and DraE (strain O75) were expressed separately as N-terminal

hexahistidine fusions in E. coli strain M15 (Qiagen) as previously described (11). Briefly, the

adhesins were purified from the supernatant using nickel affinity chromatography, followed by

size exclusion chromatography in an S75 sepharose column (Pharmacia Biotech) to separate the

trimeric forms from small amounts of monomeric, dimeric and aggregated forms of the adhesins.

CD55 was purified and refolded from E. coli also as previously described (31). AfaE-dsc, DraE-

dsc and the mutants of the two were expressed from pRSETA plasmid in BL21(DE3) cells. They

were purified via an N-terminal hexahistidine tag using Ni-NTA superflow resin (Qiagen) as

previously described for AfaE-dsc (11,30).

Chloramphenicol Sensitivity Assayed Using Surface Plasmon Resonance—Native AfaE-

III and DraE were covalently coupled to the carboxylated dextran matrix on the surface on an

acitvated CM5 sensor chip using the primary amine coupling kit (BIAcore AB). After activation

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according to the standard protocol, 0.5 mg/ml of DraE or AfaE-III in 10 mM sodium acetate pH

4.5 was injected. Differing levels were immobilized (~8,000 RU of AfaE-III and ~3,500 of

DraE) by varying the volume of protein injected. All interaction sensorgrams were collected at 20

°C by flowing 80 µl (at 20 µl/min) of a CD55 / Cm mixture where the CD55 concentration was 1

µM and the Cm concentration varied between 0 and 3.8 mM. Measurements were taken as

triplets where the first injection was of CD55 with no Cm added, the second contained Cm at the

specified concentration and the third contained Cm only. The maximum response obtained was

corrected for bulk effects (using data from a mock-coupled channel where no AfaE-III or DraE

were coupled) and non-specific chloramphenicol binding (using the signal from the Cm-only

injections). Data are plotted by taking the maximum response obtained with and without Cm and

reporting the signal as a percentage of that obtained without drug. Data presented are the mean

and standard deviations of three independent repeats.

Crystallization and Structure Determination—Crystals of AfaE-III and DraE were grown

at 21°C using sitting drop vapour diffusion. AfaE-III was concentrated to 3.5 mg/ml, while DraE

was concentrated to 1.5 mg/ml. AfaE-III-Se-Met crystals (I432) grew against a reservoir solution

containing 2 M ammonium sulphate, 2% polyethylene glycol 400 and 0.1 M sodium HEPES (pH

6.8-7.0). Native AfaE-III crystals (P3121) grew against a reservoir of 0.2 M magnesium sulphate,

20-21% polyethylene glycol 4000 and 0.1 M Tris-HCl (pH 7.8). DraE crystals (P212121) were

obtained against a reservoir solution of 2 M ammonium sulphate, 0.1 M Tris-HCl (pH 7). Co-

crystals of the DraE-Cm complex (P3) grew against a reservoir of 1.7 M ammonium sulphate,

100 µM Cm (as reported in biological assays (22) or 2.8 mM Cm) and 0.1 M NaHEPES pH 7.

All crystals were flash frozen in nitrogen gas and data collected at 100 K with crystals in a fibre

loop. Data were collected in-house, at the European Synchrotron Radiation Facility (ESRF);

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(BM-14), Grenoble and at the Synchrotron Radiation Source (SRS) (stations 9.6 and 14.2),

Daresbury.

The AfaE-III-Se-Met structure (PDB ID 1USZ) was solved at 3.1 Å by Single-

wavelength Anomalous Dispersion (SAD) with the suite of programs autoSHARP (32) and built

(as all the other structures in this paper) with the program Xtalview (33). The Se-Met model was

used to solve the AfaE-III native crystal structure by molecular replacement with the program

Molrep (34) (3.3 Å resolution, PDB ID 1UT2). The native DraE structure (1.7 Å resolution, PDB

ID 1UT1) was also solved with Molrep solved using PDB ID 1USZ as a search model. Finally,

the DraE-Cm complex (1.9 Å resolution, PDB ID 1USQ) was again solved by molecular

replacement with Molrep, using the coordinates of the native DraE as the search model. For the

DraE-Cm complex rebuilding and refinement proceded with simple addition of water and

remodelling of protein side-chains until no difference density could be identified in the Fo-Fc

map other than that associated with the Cm, at which point the model for the Cm molecule was

built.

Chemical shift mapping— 15N-labelled NMR samples of AfaE-dsc, DraE-dsc and mutants

were prepared to a final concentration of 1mM and a volume of 500µl in 50mM acetate buffer

pH5.0, 100mM sodium chloride and 10% D2O. Samples were quantified using UV absorbance at

280nm wavelength. Chloramphenicol was prepared up to a concentration of 7mM in the same

buffer as the protein and solubilized by mild heating. Chloramphenicol was added to each

sample in increments described in the text. All experiments were carried out on a 500MHz four-

channel Bruker DRX500 spectrometer equipped with a z-shielded gradient triple resonance

cryoprobe.

Coordinates and figure preparation—Coordinates and X-ray crystallographic data for the

structures have been deposited at the Protein Databank under the accession codes 1USQ, 1USZ,

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1UT1 and 1UT2. Figures were prepared using program AESOP (Martin Noble, unpublished

program).

RESULTS AND DISCUSSION

X-ray structures for oligomeric forms of AfaE-III and DraE—Whilst earlier structural

work (11) used a form of AfaE-III engineered to be monomeric (AfaE-dsc), heterologous

expression of native AfaE-III and DraE sequences in E. coli lacking the Dr fimbrial assembly

machinery leads to the production of oligomeric forms of the adhesins. These oligomers have

been extensively studied (e.g.(12,35,36)) and have been seen to possess the biological functions

of the assembled adhesins (e.g. ability to bind host cell receptors, inhibition profiles, antigenic

properties) with the exception that they do not assemble to form fimbrae. We therefore sought to

determine the structures of these oligomeric forms to facilitate comparisons with the structure of

AfaE-dsc and interpretation of existing biological data. Size exclusion chromatography (37) has

previously shown that the majority of the soluble oligomers are likely to be trimers and once

purified, trimeric oligomers of AfaE-III and DraE readily produced crystals suitable for structure

determination. The structure of an AfaE-III trimer was solved at a resolution of 3.3 Å using

multiple anomalous dispersion methods and a selenomethionine labelled form of AfaE-III (Table

1, Figure 1A and methods). The structure of DraE was then solved using this model as a starting

model for phasing by molecular replacement. In all three differently-packed crystal forms have

been obtained (two AfaE-III forms and a single DraE form), with the exception of the naturally

differing side-chains derived from the sequence differences, AfaE-III and DraE possess identical

structures (Figure 1B); the structures superimpose with an RMSD of 0.6 Å over all backbone

atoms. Moreover, structural comparison reveals a close superimposition between the average

solution structure of AfaE-dsc (11) and the crystal structures of AfaE-III and DraE monomers i.e.

an RMSD of 2.2Å over the 135 equivalent residues (Figure 1B).

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Despite possessing an identical fold to monomeric AfaE-dsc, the trimer (Figure 1A)

consists of intermolecular disulphide bonded monomers in which the N-terminal donor-strand is

folded back of to form an intramolecular, anti-parallel β-strand G. This is facilitated by

rearrangement of only five residues in the A1-A2 linker that allows β-strand A1 to slot into an

identical position in a neighbouring subunit (Figure 1A & 1C). We believe this to be an

artifactual domain-swapping that occurs as a result of cytoplasmic expression in the absence of

the chaperone and illustrates the potential problems that can occur in unassisted assembly of

fimbrae / pilins. It also represents one of only a few observations describing cyclic domain-

swapped trimers (38,39) and reflects the inherent conformational malleability of these domains.

Non-reducing SDS PAGE (Figure 1D) confirms this conclusion as analysis of adhesive

organelles purified from the surface of Dr+ bacteria could not detect the existence of a disulphide

bonded polymer. Despite this misfolding, the atomic structures of the trimeric adhesins differ

from the correctly folded form at only five residues (those in the A1-A2 linker) since, for the

majority of the residues, it is simply which subunit they originate from that differs rather than

their location within the protein fold being changed. It is therefore not surprising that earlier

biological analyses using oligomeric adhesins found them to be biologically active (12,35,36).

X-ray structure of a DraE-chloramphenicol complex—The high degree of structural

similarity between AfaE-III and DraE does not facilitate an understanding of their subtly

different biology; in particular, the precise structural basis of the differential susceptibility to

inhibition of CD55-binding by chloramphenicol (Cm) (22). We therefore decided to determine

the structure of a DraE-Cm complex. High resolution data for the DraE-Cm complex (1.9 Å,

Table 1) were collected from co-crystals grown in the presence of 2.8 mM Cm (See Experimental

Procedures). These crystals were in space group P3 and were phased by molecular replacement

with the DraE coordinates described above (See Experimental Procedures). Calculation of

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unbiased Fo-Fc difference Fourier maps prior to construction of a model for the bound Cm (Fig

2A) allowed unambiguous positioning of the drug and the coordinates for the complex were

subject to further rounds of refinement to yield the model described in Table 1 (Figure 2). Other

data sets collected from crystals that were grown in the absence of Cm and then soaked in Cm at

a range of concentrations (down to 0.1 mM) prior to data collection showed Cm to be bound in

identical fashion (data not shown). The DraE-Cm complex structure reveals that Cm binds in a

surface pocket between the N-terminal portion of strand B and the C-terminal portion of strand E,

and lies within the CD55-binding site identified by chemical shift mapping (11). Cm is anchored

at one end by van der Waals contacts of its CHCl2 moiety with the methylene groups of Gly (113

& 42) and Pro (40 & 43) residues, and at the other end by stacking of the p-nitro group against

the side-chain of Ile111 (Figure 2). The ring of Tyr115 closes the bottom of this hydrophobic

pocket with contacts to the Cl atoms and additional contacts to the Cα atoms of Ile114. Binding

of Cm in this site clearly suggests that the Cm sensitivity of DraE is due to a direct disruption of

the CD55-binding surface of DraE since the Cm masks a portion of the DraE surface previously

shown to be involved in binding of CD55 (11).

This Cm binding site is seen to be in close proximity to two of the three sequence

differences between AfaE-III and DraE (Figure 3A) and may therefore provide an explanation for

the reported lack of activity for Cm against AfaE-III binding of CD55. In the crystal structure of

AfaE-III, the Thr88Met mutation puts the side chain of Met88 into the hydrophobic pocket

occupied by Cm in DraE (Figure 3B), and the Ile111Thr mutation replaces the Ile, which packs

against the p-nitro group and neighbouring residues crucial for Cm recognition. These structural

differences seem sufficient to produce the reported lack of Cm sensitivity for AfaE-III. Carnoy

and Moseley have previously demonstrated that the single Ile111Thr mutant completely

abolishes Cm binding (22). Our data suggest that the Thr88Met mutation may also play an

important role in the lack of Cm binding to AfaE-III.

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To provide additional evidence for the observations seen in the crystal structure of the

DraE-Cm complex, we performed an NMR chemical shift mapping experiment on monomeric

DraE-dsc in the presence of increasing concentrations of Cm. A number of HSQC peaks shift

upon titration with Cm which are consistent with a weak but specific interaction between Cm and

DraE (Figure 2C). Based on changes in 15N against 1H ppm, significant peak shifts were grouped

into large, medium and small. The largest chemical shift changes were seen in amide peaks

corresponding to residues Y115 and I114, both of which are implicated by our crystal structure to

be crucial for the DraE-Cm interaction. Furthermore, the medium and small peak shifts map to

the region of the DraE-Cm interface (Figures 2D and 2E). These observations were further

probed with surface plasmon resonance experiments to examine CD55-binding by the adhesins in

the presence of Cm (Figure 3C). These studies confirm earlier reports in that CD55 binding by

DraE is seen to be Cm sensitive, with ~50% inhibition seen to occur with 2.8 mM Cm whilst

AfaE-III binding to CD55 is not significantly altered by Cm at this concentration. These results

confirm that the subtle alterations seen between the AfaE-III and DraE are indeed sufficient to

ablate Cm sensitivity.

The structure of the DraE-Cm complex may provide the start-point for the design of novel

antimicrobial compounds since, in contrast to earlier structures for Cm-ligand complexes (40,41)

where binding of Cm involves burial of the benzene ring, the interactions seen here are focussed

on burial of the chlorines. This observation may allow design of novel therapeutics that maintain

the geometry of the adhesin-Cm interaction without the nitro-benzene ring and hence avoid the

toxic side-effects associated with therapeutic use of Cm caused by intercalation into the human

ribosome. Since the lack of sensitivity to Cm of the AfaE-III is seen to be dependent on covering

of the Cm site by a single side chain we speculate that agents designed to bind with higher

affinity would be able to displace this side chain and so achieve more wide-ranging action against

this whole class of bacterial adhesins.

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Acknowledgements—The authors are indebted for the financial support of The Wellcome

Trust (Research Leave Award to SJM), BBSRC (43/B16601 to SML), arc (L0534 to SML),

MRC (Studentship to DP), EPSRC (Studentship to KLA) and the NIH (AI23598 to MEM).

Thanks to Martin Walsh at BM14, ESRF, and to the staff of stations 9.6 and 14.2 at the SRS,

Daresbury for assistance with data collection.

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39. Liu, Y., Gotte, G., Libonati, M., and Eisenberg, D. (2002) Protein Science 11, 371-380 40. Hansen, J. L., Moore, P. B., and Steitz, T. A. (2003) Journal of Molecular Biology 330,

1061-1075 41. Izard, T., and Ellis, J. (2000) Embo Journal 19, 2690-2700

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Chloramphenicol binding by DraE

Page 14

TABLE 1

Structural Statistics

a overall (outer resolution shell) b All crystals contain disulfide bonded trimers sited on either crystallographic or non-crystallographic three-fold axes.

AfaE-III trimer (1UT2)

AfaE-III trimer SeMet (1USZ)

DraE trimer (1UT1)

DraE trimer/Cm(1USQ)

X-ray Data Statistics Space Group P3121 I432 P212121 P3

Cell dimensions (Å) a=b=113.3, c=164.1 a=b=c=141.3

a=68.9, b=108.5, c=119.6

a=b=119.0, c=57.4

Resolution (Å) a 63-3.3. (3.5-3.3)

50-3.1 (3.3-3.1)

54-1.7 (1.8-1.7) 15-1.9 (2.0-1.9)

Number of unique reflections (redundancy) a 17875 (4) 5108 (23) 87802 (4) 71538 (6)

Completeness (%)a 99.9 (99.9) 100 (100) 95.7 (77.2) 99.8 (99.8) Rmergea 0.19 (0.33) 0.08 (0.32) 0.05 (0.25) 0.11 (0.33) Average I/�Ia 3.2 (2.0) 7.8 (1.7) 9.6 (3.0) 4.2 (1.4) No. Molecules in AUb 9 1 6 6 Refinement Statistics R 22.6 (24.8) 21.8 (22.9) 17.4 (21.6) 19.1 (20.2) Rfree 26.5 (26.8) 24.7 (26.0) 21.1 (23.7) 22.5 (24.0) RMSD idealized covalent geometry Bonds 0.004 0.006 0.014 0.009 Angles 0.6 0.7 1.5 0.9 Number of Atoms in model Number of Protein Atoms 9497 1059 6457 6329 Number of Non-Protein Atoms 42 10 1030 950 Mean B Protein 10 40 19 16 Mean B Non-protein 21 50 44 24 # of Sulphates / Ethylene Glycols 3 / 0 1 / 0 12 / 30 14 / 11 Ramachandran Plot Residues in most favoured regions (%) 79.1 81.7 87.1 86.0

Residues in additionally allowed regions (%) 20.4 18.3 12.9 14.0

Residues in generously allowed regions (%) 0.4 0.0 0.0 0.0

Residues in disallowed regions (%) 0.0 0.0 0.0 0.0

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Chloramphenicol binding by DraE

Page 15

Figure Captions

Figure 1 Crystallographic structures for DraE and AfaE-III, comparisons to earlier structure

of AfaE-dsc.

(A) (i) Crystal structure of AfaE-III trimer shown as a ribbon representation. Monomers are

coloured red, green and blue. The G strand that is donated from another monomer to

construct the native fimbrae (11) is striped and the five residues that differ significantly

in their place in the fold between the trimeric and monomerised forms of the protein are

highlighted in purple. (ii) ribbon representation of monomers of AfaE either taken from

the trimer (AfaE-III) or from the engineered monomer (AfaE-dsc) (11) oriented and

coloured to aid comparisons of the structures.

(B) Overlay of worm representations of the backbones of monomers from the AfaE-III

(green) and DraE (cyan) trimers. Side chains are shown for the three amino acids that

differ in sequence between the two adhesins.

(C) Topology diagrams for the adhesin trimers and for the assembled fimbrae (11).

Colouring as used in (A)

(D) Non-reducing SDS-PAGE for the fimbrial preparation of the Dr hemagglutinin (lanes 2

and 3). Markers are shown in lanes 1 and 4 and correspond to molecular weights X Y

and Z.

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Chloramphenicol binding by DraE

Page 16

Figure 2 Chloramphenicol binding to DraE

(A) Unbiased Fo-Fc difference Fourier maps calculated using data in the range 15

Å to 1.9 Å contoured at 1.9 σ. Fc was calculated from the protein model only

(before modeling of the Cm molecule). The final refined models for protein

and chloramphenicol are shown.

(B) A representation of the residues in van der Waals contact with Cm.

(C) Region of 2D 1H-15N HSQC spectra for 15N labelled DraE-dsc (black) in

presence of Cm at the molar ratio 1:5 (yellow), 1:25 (green) and 1:100

(magenta). Examples of peaks with perturbed resonance positions are

indicated.

(D) and (E) Solvent accessible surface representations of DraE with perturbed

residues coloured in red (strong shifts), orange (medium shifts) and yellow

(small shifts). Views are related by a 60 degree rotation about the long axis of

the molecule. All residues showing a chemical shift in the presence of Cm are

labelled in order to delineate the binding surface.

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Chloramphenicol binding by DraE

Page 17

Figure 3. Analysis of lack of chloramphenicol sensitivity of AfaE-III

(A) Backbone representations of the X-ray structures of DraE (cyan) and AfaE-III

(green) monomers as in Figure 1B. The alpha-carbon positions of the three

amino acids that are altered between these different strains are highlighted as

numbered space filling balls. The chloramphenicol bound to the DraE is

shown in a ball-and-stick representation and labelled Cm.

(B) Analogous view of AfaE-III to that shown of DraE in Figure 2A. The residues

that differ between these adhesins are coloured red. The side chain of Met88 is

seen to lie across the Cm-binding pocket.

(C) Cm sensitivity of CD55 binding assayed by surface plasmon resonance (see

methods). CD55 (1µM) binding to AfaE-III and DraE in the absence (-) and

presence (+) of Cm (2.8 mM). Values presented are the mean and associated

errors derived from three (DraE) and six (AfaE-III) repeated measurements.

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GF

E88

11152 B

A1B

GC2A2 F

D

E

A1

A2

GF C2

B

ED

(a) (b)

(c) (d)

C2

C1

C

C2

C1

F

CN

E

A1

G

D

B FD"

D'

A2

N

E

A1

G

D

BD"

D'

A2

n+1

n

C2

S S

S S

CN

CN

CN

E

C2

C1 A1

G

D

B FD"

D'

A2

E

C2

C1 A1

G

D

B FD"

D'

A2

E

C2

C1 A1

G

D

B FD"

D'

A2

1

2

3

to copy 1

to copy 3

S S

S S

SS

(i) (ii)

(i) (ii)

AfaE-III AfaE-dsc

MM F F

G

Figure 1

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C10

C11

N9 O9B

O9A

C9 C8

C7

C6

C5 O5

C4

O4

C3 N2

C2

O2

C1

CL2

CL1

Pro 40

Gly 42

Pro 43

Ile 111

Gly 113

Ile 114

Tyr 115

Chloramphenicol

Pro 40

Pro 43

Ile 111

Ile 114

Gly 113

Tyr 115

Gly 42

(a) (b)

(d)

Thr 88

(c)

(e)

Tyr 115

Ile 114

Gly 42Ile 41

Tyr 115

Ile 114

Ile 41

Gly 42

Val 116

Ala 38

Lys 113

Val 46

Lys 24Thr 10

Ser 5

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(a)

(b)

(c)

Thr 111

Met88

111

88

52 Cm

G

EB

F

CD

55

bin

din

g(%

)

- +

AfaE-III

- +

DraE

0

20

40

60

80

100

120

Figure 3

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Bouguenc, Steve Matthews and Susan M. LeaUrvil, Filip Rabuzin, Pietro Roversi, Bogdan Nowicki, Laurence du Merle, Chantal Le

David Pettigrew, Kirstine L. Anderson, Jason Billington, Ernesto Cota, Peter Simpson, Petrichloramphenicol

High resolution studies of the Afa/Dr adhesin DraE and its interaction with

published online August 24, 2004J. Biol. Chem. 

  10.1074/jbc.M409284200Access the most updated version of this article at doi:

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