Study of the Structural and Dynamic E!ects in the FimH Adhesinupon !!D!Heptyl Mannose BindingSophie Vanwetswinkel,†,‡ Alexander N. Volkov,*,†,‡ Yann G. J. Sterckx,†,‡ Abel Garcia-Pino,†,‡

Lieven Buts,†,‡ Wim F. Vranken,†,‡ Julie Bouckaert,‡,¶ Rene ! Roy,§ Lode Wyns,†,‡

and Nico A. J. van Nuland*,†,‡

†Jean Jeener NMR Centre, Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium‡Molecular Recognition Unit, Structural Biology Research Center, VIB, Pleinlaan 2, 1050 Brussels, Belgium§Department of Chemistry, Universite ! du Que !bec a "Montre !al, P.O. Box 8888, Succ. Centre-Ville, Montre !al (QC), Canada H3C 3P8

*S Supporting Information

ABSTRACT: Uropathogenic Escherichia coli cause urinary tractinfections by adhering to mannosylated receptors on the humanurothelium via the carbohydrate-binding domain of the FimH adhesin(FimHL). Numerous !-D-mannopyranosides, including !-D-heptylmannose (HM), inhibit this process by interacting with FimHL. Toestablish the molecular basis of the high-a!nity HM binding, wesolved the solution structure of the apo form and the crystal structureof the FimHL!HM complex. NMR relaxation analysis revealed thatprotein dynamics were not a"ected by the sugar binding, yet HMaddition promoted protein dimerization, which was further con#rmedby small-angle X-ray scattering. Finally, to address the role of Y48, partof the “tyrosine gate” believed to govern the a!nity and speci#city ofmannoside binding, we characterized the FimHL Y48A mutant, whoseconformational, dynamical, and HM binding properties were found tobe very similar to those of the wild-type protein.

! INTRODUCTIONLocated at the tips of the type-1 pili of uropathogenicEscherichia coli (UPEC) strains, the FimH adhesin is themost prevalent causative agent of human urinary tractinfections (UTIs). Bacterial invasion is e"ectively mediatedby the adhesion of FimH to mannosylated receptors on theurinary epithelium through its amino-terminal, carbohydrate-binding domain, referred to as lectin domain (FimHL). Thecarboxy-terminal pilin domain of FimH (FimHP) anchors thewhole protein to the pili rod on the bacterial surface. In thenative context of the #mbriae, both domains are interdepend-ent, and the sugar a!nity is allosterically regulated through amolecular mechanism established in an elegant study byLeTrong and colleagues.1 Brie$y, FimHP makes internalcontacts with FimHL causing the loosening of the mannose-binding pocket and thereby maintaining FimH in a low-a!nitystate. Upon separation of the domains through sugarinteraction and/or tensile forces, the FimHL conformationrearranges toward a high-a!nity state featuring a tighterbinding site.Because of their frequency of occurrence, UTIs represent an

important public health issue. Women are particularly a"ected,as almost half of them will experience at least one infection atsome point in their lives. Moreover, despite the currentavailability of antibiotic treatments, UTIs recur two or more

times within months of a primary infection in 20!30% of thepatients and evolve into a chronic form in about 5% of thecases.2 Combined with the threat of increasing antibioticresistance among bacterial strains, this context has been fuelingthe research toward the discovery of e!cient FimH antagonistswith therapeutical value. Most of the reported studies aiming atdesigning and optimizing new drugs have been performed onthe isolated FimHL domain displaying the high-a!nityconformation.2!6 Indeed, the latter is small and easy toproduce as opposed to the full-length protein, which is prone toaggregation due to the incomplete Ig-fold of FimHP lacking a "-strand donated by the FimG adaptor subunit in the mature#mbriae.1

Since the discovery that aryl !-D-mannosides were potentinhibitors of FimH-mediated bacterial adhesion,7 a lot of e"orthas been directed toward the development of new syntheticderivatives with improved antiadhesive properties. An im-portant step in this direction was made in a study reporting thecrystal structure of FimHL bound to butyl mannose anddemonstrating that alkyl mannosides act as high-a!nity(nanomolar) FimH ligands,8 which established the structuralbasis of sugar binding. The subsequent crystal structure of

Received: October 28, 2013Published: January 29, 2014

Article

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FimHL in complex with oligomannose-3, a physiological ligandmimic, con#rmed that the binding was mostly driven by asingle sugar ring embedded in a deep, polar monosaccharidebinding pocket making the most of the protein contacts.9 Thiswork thereby validated the earlier idea that monomannosideswere su!cient for sugar recognition and tight binding andcould act as potent competitive inhibitors of the naturalligand.10,11

Since then, synthetic monomannosides carrying variousaglycone chains have been reported and their binding toFimHL has been characterized. Several crystal structures ofFimHL!monomannoside complexes are available,2,4 illustratingthat, as in the case of butyl mannose, the aglycone interacts viavan der Waals contacts and/or aromatic stacking with ahydrophobic collar surrounding the binding pocket. Morespeci#cally, the interaction is mediated by the residues Tyr48,Tyr137, and Ile52, forming the so-called “tyrosine gate”. In awork combining structural and thermodynamic studies, Wellenset al. suggested that changes in the orientation and dynamics ofthese residues extending from the binding pocket, in particularthe Tyr48, could govern the binding a!nity and speci#city ofsynthetic antagonists.2

To address the role of the tyrosine gate in more detail, wehave studied FimHL by NMR spectroscopy and obtained thesolution structure of the apo form. We investigated thestructural and dynamics e"ects of heptyl mannose (HM)binding in solution by isothermal titration calorimetry (ITC),nuclear magnetic resonance (NMR) spectroscopy, and small-angle X-ray scattering (SAXS). Moreover, we solved the crystalstructure of the FimHL!HM complex, for which there were noatomic details available so far. In parallel, the Y48A FimHLmutant was prepared and its binding properties and dynamicswere compared with that of the wild-type (wt) protein.

! RESULTS AND DISCUSSIONStructure and Dynamics of the Apo FimHL. The good

peak dispersion in the [1H,15N]-heteronuclear single-quantumcoherence (HSQC) spectrum (Figure S1, Supporting In-formation) and high quality of the triple-resonance dataenabled nearly complete assignment (backbone 96.5%, side-chain 1H 97.6%, side-chain non-1H 84.3%, BMRB entry 19066)of the apo wild-type (wt) FimHL

1H, 13C, and 15N nuclei,including all non-proline backbone amides.The NMR structure of the wt FimHL in its apo form was

solved using NOE-derived distance restraints and a combina-tion of dihedral angle restraints. All unambiguous distancerestraints were generated with CYANA12,13 based on theautomatic assignment of the 3D NOE cross-peaks. About 80%of the NOEs were unambiguously assigned, leading to anaverage of 29.2 NOE restraints per residue; the remainingincompleteness is most likely due to the few missing chemicalshift assignments and a semiautomated peak-picking procedureapplied to crowded spectra. In addition to the unambiguousrestraints, nine distance restraints were added manually toaccount for the ambiguity of the Phe1 aromatic protonassignments. Indeed, these protons were tentatively assignedbased on the 2D NOESY and 3D 13C-NOESY-HSQC spectra,where the ambiguity could not be resolved. In addition, thePhe1 H# and H$ resonances could not be identi#ed in the 2D(HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE experi-ments. Omitting these additional restraints during the structurecalculation resulted in an unrealistic Phe1 conformation fullyexposed to the solvent.

The ensemble of the 25 water-re#ned lowest-energy FimHLstructures (Figure 1A) is in excellent agreement with the

experimental data showing no signi#cant distance violations.The satisfactory red-orange-green (ROG) CING validationscore14 and Ramachandran statistics (Table S1, SupportingInformation) further illustrate the good quality of the solutionstructure. The atomic coordinates were deposited to theProtein Data Bank under the accession number 3zpd.The solution structure was obtained in the absence of any

ligand, whereas, to the best of our knowledge, all crystalstructures reported so far for the isolated FimHL domaincontain either mannose, synthetic mannosides, or ethyleneglycol, mimicking the O2, O3, and O5 hydroxyls of !-D-mannopyranose,2 at the sugar binding site. Interestingly, in thesolution structure of the apo FimHL, the tyrosine 48, which hasbeen proposed to govern the sugar binding a!nity andspeci#city,2 adopts an “open” conformation similar to the oneobserved in the X-ray structure of the complex witholigomannose-3,9 a natural substrate analogue (Figure 1B).Overall, the NMR structure of the apo FimHL and crystalstructures of the FimHL!ligand complexes are very similar,with an average backbone root-mean-square deviation (RMSD)of 1.44 Å (Table S2, Supporting Information). Yet, while thesecondary structure elements superimpose well, several loops,especially those located near the binding site and appearingwell-de#ned in the crystal structures (Figure S2, SupportingInformation), display di"erent conformations. It is noteworthythat the NMR ensemble shows higher variability for theseregions compared to the rest of the protein (Figure 1A), mostlikely due to smaller number of NOE distance restraintsobtained for these residues (Figure S3, Supporting Informa-tion).In principle, such structural heterogeneity could arise from

local loop motions; however, as discussed below, NMRrelaxation analysis reveals no signi#cant protein dynamics at

Figure 1. Solution structure of the apo wt FimHL. (A) Ensemble ofthe 25 lowest energy structures represented as ribbons. (B) Overlay ofthe cartoon representation of the model closest to the mean (in blue)with the crystal structure of the FimHL!oligomannose-3 complex(PDB 2VCO; the protein is in gray and oligommanose-3 in orange).The tyrosine 48 (in open conformation) is shown in sticks. The insetshows the sugar binding site residues Y48, I52, and Y137 forming the“tyrosine gate”. Molecular graphics were generated with PyMOL15 andChimera.16

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the backbone level and shows that FimHL is essentially rigid onthe ps!ns and %s!ms time scales. Given that HM binding toFimHL in solution a"ects only the sugar binding site residues(see below), we believe that the di"erences observed for theapo and holo FimHL structures re$ect the experimentaldiscrepancies between the solution NMR spectroscopy andX-ray crystallography, rather than conformational changesinduced by the sugar binding. Indeed, in FimHL-ligand X-raystructures, the loops surrounding the binding site are oftenengaged in intermolecular contacts dictated by the crystallattice, which might explain the di"erence in conformationscompared to the NMR structure determined in this work.Except for several isolated outliers and slightly increased R2

rates for the residues 135!141, the apo wt FimHL exhibits $atR1 and R2 relaxation pro#les (Figure S4, SupportingInformation), indicating the absence of signi#cant backbonedynamics on the ps!ns time scale. Furthermore, backboneamides of the apo wt FimHL show $at R2 relaxation dispersionpro#les, implying that either the protein undergoes noconformational exchange on the %s!ms time scale or thedi"erence in chemical shifts between the exchanging species issmall.17 Finally, a single set of well-resolved peaks in the HSQCspectra signi#es the presence of a single protein species on theNMR chemical shift time scale (ms!s). Taken together, theNMR relaxation data demonstrate the absence of protein

backbone dynamics on the ps!ns and %s!s time scales.Although the present experiments did not address the motionson the intermediate, ns!%s time scale, a time regime not easilyaccessible to the solution NMR spectroscopy,17 given theprotein backbone rigidity on either side of this time-window,large-scale ns!%s protein dynamics appear to be rather unlikely.Being sensitive to the shape of the protein, the R2/R1 ratio

can be used to determine the hydrodynamic properties of themolecule.18 The analysis of the R2/R1 data shows that thedi"usion tensor of the apo wt FimHL is accurately described byan axially symmetric particle with Dpar/Dper = 1.46 ± 0.02 and arotational correlation time tc = 11.2 ns. Though the absolutevalue should be taken with caution due to the self-associationpropensity of FimHL (see below), the latter value is in a goodagreement with &c = 10.1 ns obtained from the structure-basedhydrodynamic calculations (see Experimental Section).The behavior of the apo FimHL in solution was also

investigated by SAXS under the conditions similar to those ofthe NMR experiments (Figure 2A). The #nal scattering curve is#tted well by the apo FimHL solution structure, and ab initioshape reconstruction adequately produces a model thatmatches the protein monomer (Figure 2B). Thus, the SAXSanalysis con#rms the validity of the apo FimHL NMR structure.

Structural and Thermodynamic Characterization ofthe wt FimHL!HM Interaction. HM is routinely used as a

Figure 2. SAXS analysis for the apo and HM-bound FimHL. Bu"er-subtracted scattering curves of (A) apo and (C) holo FimHL measured at fourdi"erent concentrations: 2.0, 4.0, 6.0, and 7.5 mg/mL. The curves are shown in di"erent shades of blue and green for apo and holo FimHL,respectively. In both cases, the curves are displaced vertically relative to each other and are plotted on an arbitrary intensity scale for clarity. The graytraces depict the experimental error. The insets show the Guinier regions of the respective scattering curves. (B) The merged scattering curve for apoFimHL (black dots, gray traces depict the experimental error) is very well described by an apo FimH monomer. The red and blue lines represent theCRYSOL #t for the apo FimHL NMR structure and the #t for the best ab initio model, respectively. An overlay of the apo FimHL NMR structure(blue ribbon) and the ab initio model (gray beads) is shown on the right. (D) The percentages of holo FimHL monomer and dimer as a function ofconcentration obtained by a linear combination of scattering from the dimer and monomer by OLIGOMER ('2 in black). Given for comparison, the'2 in red correspond to the #ts with 100% of monomer. The #ts of the di"erent OLIGOMER runs are shown as red traces in C.

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reference in drug discovery programs because it is easilyaccessible, is water-soluble, and remains one of the strongestmonovalent FimHL binders described so far.2!6 However, inthe absence of structural data, the reasons for this high a!nityremained subject to speculation.2 Here, we obtained the crystalstructure of the wt FimHL!HM complex at 1.6 Å resolution(Figure 3A; Table S3, Supporting Information). Clear electron

density was observed for HM, including its aglycone tail, whichappeared to be well ordered. With a 0.11 Å backbone rmsd, thestructure features virtually identical C! traces compared to thebutyl mannose-bound FimHL.

8 As in the latter, the Tyr48 isfound to be in the closed conformation. The ligands in bothstructures are also highly superimposable, with their alkylmoieties extending out of the mannose-binding pocket towardthe hydrophobic patch formed by Tyr48, Tyr137, and Ile52.However, while this patch is only partially covered in the caseof butyl mannose, the HM aglycone tail (longer by threecarbon atoms) allows additional van der Waals contacts, whichcould account for the "20 fold higher binding a!nity.Moreover, contrary to what was observed for the apo FimHL

solution structure, the loops surrounding the sugar binding siteof the HM-bound protein adopt conformations similar to theone seen in all previously crystallized FimHL complexes, whichcon#rms that they are well-de#ned in X-ray structures (FigureS2, Supporting Information). In the case of our FimHL!HMcomplex, these loops are de#nitely involved in crystal contacts.Indeed, the crystal packing appears to involve the formation ofhead-to-tail dimers, where the sugar binding site of onemolecule contacts the carboxy-terminal part of a second one(Figure 3B). Favorable protein!protein interactions are furtherreinforced by the HM ligand, contributing sugar!proteincontacts.

As many of the resonances in the HSQC spectra of the apoand the HM-bound wt FimHL were virtually identical, theirNMR assignments could be easily transferred from the formerspectrum to the latter. These were veri#ed, and the assignmentof the HSQC spectrum of the wt FimHL!HM was completedby triple-resonance NMR experiments (see ExperimentalSection). Signi#cant line broadening was observed upon sugaraddition, and the consequent loss of resolution resulted in alimited assignment of the theoretically observable side-chainatom resonances in the FimHL!HM complex (backbone96.3%, side-chain 1H 85.4%, side-chain non-1H 71.8%, BMRBentry 19256).Despite this line broadening, which e"ectively prevented the

calculation of the NMR structure of the HM-bound FimHLform, the analysis of the backbone amide chemical shiftperturbations indicated that, also in solution, HM binding onlya"ects the sugar binding site residues (Figure 4). Moreover, the

protein!sugar interaction was found to be in slow exchangeregime on the NMR chemical shift time scale as expected fromthe nanomolar a!nity of HM for the wt FimHL (see ITCbelow and ref 2). NMR exchange regime is de#ned by theexchange constant (kex) and the di"erence in the chemical shiftsbetween the free and bound forms (##) as fast (kex # ##),intermediate (kex $ ##), or slow (kex % ##). The latter ismanifested by the presence of two sets of peaks, correspondingto free and bound forms, at nonsaturating ligand concen-trations.

Figure 3. Cartoon representation of the crystal structure of theFimHL!HM complex. (A) Overlay of the crystal structures of FimHLbound either to HM (this work, in green) or to butyl mannose (PDB1UWF, in gray). HM and butyl mannose are shown in black and gray,respectively; the tyrosine 48 (in closed conformation) is shown insticks. (B) Head-to-tail dimer as observed in the crystal.

Figure 4. Combined backbone amide chemical shift perturbations(##avg) induced upon HM binding to the wt FimHL (A) plotted foreach observed backbone amide, and (B) mapped onto a surfacerepresentation of the FimHL!HM crystal structure (PDB 4lov, thiswork). Residues are colored by the ##avg as de#ned in the ramp.Proline and unassigned residues are shown in light gray, and HM indark gray sticks.

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Addition of HM to the wt FimHL does not change thepattern of the R1 and R2 relaxation pro#les but leads to auniform decrease in the R1 and concomitant increase in the R2rates for all FimHL backbone amides (Figure S4, SupportingInformation). This behavior indicates an increase in the overallrotational correlation time (&c) of the system, most likelyarising from partial, sugar-induced FimHL dimerization (seebelow). In a similar fashion, small systematic shifts of therelaxation pro#les are observed at a higher concentration of theapo protein (Figure S5, Supporting Information), whichsuggests that FimHL shows signs of self-association also inthe absence of the sugar, con#rming the observations of arecent report.19 Like the apo protein, FimHL!HM shows $atR2 relaxation dispersion pro#les. Overall, these #ndings suggestthat the HM binding has virtually no e"ect on the proteinbackbone dynamics. In contrast to the apo FimHL, the R2/R1data for the HM-bound protein could not be reliably analyzedwith a simple model, most likely due to the presence of severalspecies populated at the monomer!dimer equilibrium (seebelow).The binding of HM to the wt FimHL was measured by ITC

in the same conditions as used for the NMR experiments. As itis practically di!cult to accurately determine the concentrationof the sugar stock solution, and thereby the bindingstoichiometry, both direct and reverse titrations wereperformed (Figure 5). In both cases, the observed number ofbinding sites (n) was close to 1, con#rming the 1:1stoichiometry of the FimHL!HM interaction.

As expected, HM displayed a strong a!nity for the FimHL(KD " 17!19 nM), in good agreement with the previouslyreported value of "7 nM.2 Although the thermodynamicsignature is consistent with the binding being driven by arelatively large enthalpic contribution (#H " 11.6 kcal/mol),here we observed only a slight entropic loss (!T#S " 1 kcal/mol vs 2.65 kcal/mol in ref 2) (Table 1). This discrepancylikely re$ects di"erences in the (de)solvation energies of thebinding partners due to the di"erent experimental conditionsused in this and earlier works.

Analysis of the HM-Induced FimHL Monomer!DimerEquilibrium in Solution. To investigate whether a mono-mer!dimer equilibrium exists in solution upon addition of HMto FimHL, SAXS studies were carried out. Analysis of theln[I(q)] vs q2 plots (Figure 2C, inset) using Guinierapproximation ln[I(q)] = ln[I(0)] ! q2Rg

2/3 allows extractingI(0) and the Rg, which are, respectively, the forward scatteringintensity (related to the e"ective molecular weight of thescattering particle) and the apparent radius of gyration. As canbe seen from the rise of I(0) and Rg with the holo FimHLconcentration (the latter increases from 17.90 Å to 21.24 Å),the addition of HM to the FimHL causes an increase in particlesize. A similar trend is observed in the plot of the distancedistribution function, p(r) (Figure 6A), which featuresincreased occurrences of the larger particle diameters with therising protein concentration. Thus, unlike for the apo FimHL,the scattering curves for the holo FimHL show signi#cantconcentration dependence, suggesting possible sugar-inducedprotein dimerization. Indeed, in contrast to the Guinier regions

Figure 5. ITC binding curves. (A) Direct titration of HM into the wt FimHL and (B) reverse titration of the wt FimHL into HM. The top andbottom panels show, respectively, the raw data after the baseline correction and the integrated data corrected for the heat of dilution of the ligand.The solid line in the bottom panel is the best #t of the data to an n identical and independent site-binding model. See Table 1 for the measuredthermodynamic parameters.

Table 1. ITC Thermodynamic Parameters for the Binding of HM to the FimHL WT and Y48A Mutant

n Kd (nM) #H (kcal/mol) -T#S (kcal/mol) #G (kcal/mol)

wt (direct) 0.95 ± 0.01 17.1 ± 2.7 !11.59 ± 0.08 1.00 ± 0.12 !10.59 ± 0.14wt (reverse) 0.96 ± 0.01 19.2 ± 4.6 !11.65 ± 0.09 1.12 ± 0.17 !10.53 ± 0.19Y48A (direct) 1.04 ± 0.01 94.3 ± 12.9 !8.99 ± 0.08 !0.64 ± 0.11 !9.63 ± 0.14

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of the apo FimHL curves, the ones measured for the holoFimHL are not parallel (compare Figures 2A, C).If HM-induced holo FimHL dimerization is at play, it should

be possible to #t the di"erent concentration curves by the linearcombinations of the scattering of a holo FimHL monomer anddimer using OLIGOMER.20 Given that no prior informationon the shape of the putative holo FimHL dimer is available, andthat the crystallographic head-to-tail dimer could be acrystallization artifact, di"erent holo FimHL dimers weregenerated using AUTODOCK (Figure 6B).21 From the

AUTODOCK run, di"erent solutions with distinct shapeswere selected and used for #tting the scattering curve at thehighest holo FimHL concentration. As can be seen from Figure6C, only the elongated crystallographic dimer produces a good#t compared to the other, more compact, dimeric forms.However, because of the inherently low resolution of the SAXSexperiment, we cannot unambiguously de#ne the head/tailarrangement of the protein units as the head-to-tail, head-to-head, and tail-to-tail dimers provide equally good #ts of theSAXS curves (Figure 6D). Although analyzed in the context of

Figure 6. SAXS curve #tting using di"erent shape dimers for the holo FimHL. (A) p(r) functions of apo and holo FimHL support the occurrence of aconcentration-dependent dimerization in the presence of HM. (B) Di"erent AUTODOCK solutions for the holo FimHL dimer are shown in purple,yellow, and red, respectively. The crystallographic dimer is shown in green, and the HM is depicted in a sphere representation. (C) Fits ofOLIGOMER runs for the di"erent dimers shown in B to the experimental data of the highest holo FimHL concentration (black dots; gray tracesrepresent the experimental error). (D) Same as C for head-to-tail, tail-to-tail, and head-to-head dimers.

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the crystallographic dimer, the NMR relaxation data do nothelp to resolve this ambiguity as the overall shape of theassembly, which largely determines the relaxation properties ofthe individual atoms, remains the same irrespective of the head/tail orientation. Nevertheless, it is clear that the holo FimHLdimer is elongated (Figures 6C,D).Analysis of the di"erent concentration curves using the holo

FimHL monomer and the elongated (head-to-tail) dimerdemonstrates that the population of the latter increases withthe protein concentration. The lowest and highest holo FimHLconcentrations correspond, respectively, to the monomericprotein and the equilibrium mixture of the 80% monomer and20% dimer (Figure 2D). This HM-induced dimerization seemsto be an intrinsic property of the system, as performing theexperiments under di"erent bu"er conditions yields very similarresults (Figures S6 and S7, Supporting Information).To complement the SAXS experiments and characterize the

sugar-promoted FimHL dimerization under the conditions ofthe NMR experiments, we have performed the followinganalysis. First, the R2/R1 pro#les for the backbone amides of theFimHL monomer and dimer were obtained from thecorresponding atomic coordinates (#lled symbols in Figure7A). Then the sum of the population-weighted R2/R1 values ofthe individual species, (R2/R1)calcd, was calculated at varyingdimer populations (p), and the agreement between theexperimental R2/R1 and the (R2/R1)calcd was assessed at eachp value by computing a Q factor (see Experimental Section).The lower the Q factor, the better the agreement between (R2/R1)calcd and the experimental data; thus, the global minimum ofthe Q = f(p) function corresponds to the best solution for the pvalue (Figure 7B). Here, the smallest Q = 0.12 is obtained at p= 0.17, suggesting that the NMR relaxation data can be wellexplained by the presence of 17% protein dimer and 83%monomer in the FimHL!HM solution (Figure 7A). The pvalue derived from the NMR relaxation measurements (p =0.17) is in good agreement with that obtained from the SAXSexperiments conducted at the highest protein concentration (p= 0.2; see above). Thus, the present Q = f(p) analysis of the R2/R1 data complements the SAXS #ndings and provides anindependent means to assess the sugar-induced FimHLmonomer!dimer equilibrium in solution.These results are con#rmed further by NMR di"usion

measurements. Structure-based hydrodynamic calculations

indicate that the translational di"usion coe!cient, D, of thecrystallographic, HM-bound FimHL dimer is expected to be1.41 times smaller than that of the monomeric protein. Withthe D = 1.27 ± 0.01 ! 10!10 m2 s!1 measured by NMRdi"usion-ordered spectroscopy (DOSY) for the apo FimHL, aD = 0.90 ! 10!10 m2 s!1 is expected for the FimHL!HMprotein dimer. Assuming an equilibrium mixture of 17% dimerand 83% monomer as established by the NMR relaxationanalysis (see above), an overall D = 1.19 ! 10!10 m2 s!1 isexpected for the FimHL!HM system, which is indeed veryclose to the value (D = 1.20 ± 0.01 ! 10!10 m2 s!1) obtained byDOSY on the HM-bound FimHL sample. Thus, the NMRdi"usion measurements agree with the monomer!dimerequilibrium in FimHL!HM solutions as established by SAXSand NMR relaxation analyses.

Study of the FimHL Y48A Mutant. In an e"ort to furtherinvestigate the role of the tyrosine 48, we prepared a FimHLY48A mutant. The backbone amide resonances for both theapo (BMRB entry 19255) and HM-bound (BMRB entry19254) proteins were assigned by comparing the [1H,15N]-HSQC and 15N-NOESY-HSQC spectra with those of the wtprotein. Note that, in the case of the bound form, the Asp47and Asn96 backbone amide chemical shifts were not identi#ed.Chemical shift perturbation analysis con#rmed that themutation did not a"ect the overall structure of the proteinbecause its e"ects were restricted to the direct neighbors ofresidue 48 (Figure S8A, Supporting Information). Moreover,just as observed for the wt FimHL, the HM binding shifts werelimited to the sugar recognition site (Figure S8B, SupportingInformation).The a!nity of HM for the Y48A mutant was assessed by ITC

(Figure S9, Supporting Information). Compared to the wtprotein, we observed "5-fold decrease in Kd, accompanied by aslight decrease of enthalpy, partly compensated by an entropygain (Table 1). These relatively small changes in the bindingconstant and thermodynamic parameters do not support anactive role of the residue 48 in HM recognition. Rather, theycan be explained by di"erences in solvent reorganization uponbinding, because an entropy gain is generally due to watermolecules being expelled from the complex interface.22 Indeed,the replacement of the hydrophobic solvent-exposed tyrosine48 by an alanine potentially could create an additional siteoccupied by ordered water molecules.

Figure 7. NMR relaxation analysis of the wt FimHL!HM monomer!dimer equilibrium. (A) R2/R1 pro#les calculated for the apo monomericFimHL (#lled circles), head-to-tail crystallographic dimer of the HM-bound FimHL (#lled triangles), and the linear combination of the predictedvalues for the 83% monomer and 17% dimer mixture (black open circles) are compared to the experimental data (red open circles). The experimentswere conducted on a 1.08 mM wt FimHL!HM sample in 20 mM sodium phosphate/100 mM NaCl pH 6.0 at 25 °C and 600 MHz. (B) Q factor asa function of the dimer population (p). See text for details.

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For both apo and HM-bound Y48A FimHL, the R1 and R2pro#les (Figure S10, Supporting Information) and the $at R2relaxation dispersion curves are virtually identical to those ofthe wt protein, indicating that the introduced mutation neitheralters the protein backbone dynamics nor perturbs the sugar-induced FimHL monomer!dimer equilibrium.

! CONCLUDING REMARKSWe have established the molecular determinants of the high-a!nity HM binding to FimHL. Overall, our data do not supportan active role of Y48 in HM recognition and demonstrate itslimited in$uence on protein backbone dynamics. From themethodological perspective, this work illustrates the importanceof the combined use of several biophysical techniques (i.e.,SAXS, NMR relaxation, and di"usion measurements) forcomprehensive analysis of protein monomer!dimer equili-brium in solution. Our results, in particular the #ndings of thesugar-induced dimerization of the isolated FimHL domain,unlikely to occur in the context of the full-length protein, couldbene#t future studies of ligand binding to FimH and areexpected to inform the ongoing drug discovery e"orts.

! EXPERIMENTAL SECTIONHeptyl !-D-Mannopyranoside (HM). The sugar was synthesized

at the Department of Chemistry, the University of Quebec (Montre !al,Canada), through Fischer glycosidation by coupling D-mannose and 1-heptanol catalyzed with camphorsulfonic acid.23

Expression and Puri"cation of FimHL. Recombinant wt FimHLwt and its Y48A mutant were produced under the control of a T7promoter using a construct where the endogenous FimH signalpeptide was replaced with the PelB one to increase the yield ofperiplasmic export. Therefore, the gene encoding the residues 1 to 158of the FimH adhesin from the uropathogenic E. coli (UPEC) strain J96was ampli#ed from the plasmid pMMB91,9 appended with the PelBsequence and subcloned into the pET24a(+) vector (Novagen). Theprotein was expressed from the resulting construct transformed into E.coli C43 (DE3) cells24 grown in M9 minimal medium (6.8 g/LNa2HPO4, 3 g/L KH2PO4, 1 g/L NaCl) containing 25 mg/Lkanamycin and supplemented with 2 mM MgSO4, 0.2 mM CaCl2,trace elements (60 mg/L FeSO4·7H2O, 12 mg/L MnCl2·4H2O, 8 mg/L CoCl2·6H2O, 7 mg/L ZnSO4·7H2O, 3 mg/L CuCl2·2H2O, 0.2 mg/L H3BO3, and 50 mg/L EDTA), BME vitamin mix (Sigma), and 1 g/LNH4Cl plus 4 g/L D-glucose. Bacteria were grown at 37 °C tillOD600 nm reached 0.6!0.8. At this point, the protein expression wasinduced with 1 mM IPTG, and the culture temperature was lowered to30 °C. After overnight incubation, the cells were harvested bycentrifugation, and the bacterial pellets were resuspended in 30 mMTris-HCl pH 8.0 bu"er containing 20% sucrose and stored at !80 °C.The same procedure was applied to the expression of the uniformly

labeled [15N] and [13C, 15N] proteins, except that 1 g/L 15NH4Cl(CortecNet) and 2 g/L 13C6-glucose (Cambridge Isotope Laborato-ries) were used as the sole nitrogen and carbon sources.To purify the FimHL domain, the frozen cells were thawed, 1 mM

EDTA and protease inhibitors were added (0.1 mg/mL AEBSF-HCland 1 mg/mL leupeptin, Roche), and periplasmic extracts wereprepared. For this, the thawed bacterial suspensions were cleared bycentrifugation (12 000 rpm for 15 min), the supernatant (sucrosefraction) kept on ice, and the pellet resuspended in ice-cold 5 mMMgSO4. After incubation of the latter at room temperature and slightagitation for 10!15 min, the solution was spun down (20 000 rpm for20 min), and the supernatant was pooled with the sucrose fraction toobtain the total periplasmic extract, which was dialyzed against 20 mMsodium acetate pH 4.0 at 4 °C for 3 to 4 h. The sample was harvestedand cleared by centrifugation, and the pH and conductivity werechecked and, if necessary, adjusted by dilution with water to pH 4.0and &1 mS/cm, respectively.

The periplasmic extract was loaded onto a 10 mL Source 30Scolumn (GE Healthcare) pre-equilibrated in 20 mM sodium acetatepH 4.0. After washing with 20!30 mL of the equilibration bu"er, theprotein was eluted with a 100!150 mL gradient of 0!500 mM NaClin 20 mM sodium acetate pH 4.0. The protein-containing fractionswere directly neutralized with 1 M Tris-HCl pH 8.0, pooled,exchanged into the required bu"er, and then concentrated to 5!20mg/mL in a Hydrosart centrifugal device (5000 cuto", Vivaspin).

The Y48A mutation was introduced into the pET24a(+)-derivedFimHL expression plasmid via whole plasmid synthesis polymerasechain reaction.25 The Y48A FimHL variant was produced and puri#edas described for the wt protein.

As they were prepared from cultures in M9 minimal medium, all theprotein samples (labeled and not) used in this study were in the apoform. The samples were found to be very stable (upon at least 6month storage at 4 °C) at pH 6.0 and under relatively low ionicstrength (20 mM sodium phosphate containing 100 mM NaCl), bu"erconditions ideal for biomolecular NMR spectroscopy. Proteins wereroutinely stored in a cold-room or kept at !80 °C for longer-termstorage.

NMR Chemical Shift Assignments. NMR experiments wereperformed at 298 K (unless mentioned otherwise) either in-house onVarian NMR Direct-Drive Systems 600 and 800 MHz spectrometers,the latter equipped with a salt tolerant 13C-enhanced PFG-Z coldprobe, or at the University of Utrecht (BIO-NMR partner, TheNetherlands) on Bruker Avance II 900 MHz and Bruker Avance DRX600 MHz spectrometers, both equipped with 5 mm TCI cryo HCN Z-GRD cryogenic probes. All NMR data were processed in NMRPipe26

and analyzed in CCPN.27

A set of 2D [1H,15N]-HSQC, [1H,13C]-HSQC, 3D NOESYs [15N-NOESY-HSQC, and 13C-NOESY-HSQC for aliphatics and aromatics,mixing time 100 ms] and assignment spectra [CBCA(CO)NH,HNCACB, HNCO, HBHA(CO)NH, C(CO)NH, and HCCH-TOCSY28 as well as (HB)CB-(CGCD)HD and (HB)CB(CGCDCE)-HE for aromatics29] was #rst recorded for the apo form on a samplecontaining 1 mM [13C, 15N] double-labeled FimHL in 20 mM sodiumphosphate/100 mM NaCl pH 6.0 and 6% D2O for the lock. Then HMwas added to the sample till saturation, and the same suite ofexperiments was acquired for the sugar-bound form. For completion,2D NOESY spectra were also recorded for both forms using unlabeledFimHL (0.5 mM) resuspended in D2O after lyophilization of a proteinsolution in 20 mM sodium phosphate/100 mM NaCl pH 6.0. Samplescontaining 1 mM of apo and HM-bound [15N] labeled Y48A in 20mM sodium phosphate/100 mM NaCl pH 6.0/6% D2O were used forthe acquisition of [1H,15N]-HSQC and 15N-NOESY-HSQC spectra .

Nearly complete H, N, and C nuclei assignment was obtained forthe apo FimHL, and above 80% of these resonances were determinedfor the HM-bound form following a standard procedure.30 Sequentialbackbone assignments were obtained by connecting 13Ca and 13Cbfrequencies from the HNCACB and CBCA(CO)NH spectra at the1H, 15N frequencies of every peak in the [1H,15N]-HSQC spectrum.Subsequently, 1H!, 1H", and side-chain 13C chemical shifts wereobtained from the HBHA(CO)NH and C(CO)NH spectra,respectively. The de#ned 1H!!13C! and 1H"!13C" resonanceswere then used for the assignment of the 1H and missing 13Cfrequencies of aliphatic side chains from the HCCH-TOCSYspectrum, and the 13CO resonances were deduced from the HNCOspectrum. Aromatic 1H chemical shifts were obtained from thecombined use of the (HB)CB(CGCD)HD and (HB)CB(CGCDCE)-HE spectra, based on the correlation with the assigned 13C"resonances; the corresponding aromatic 13C frequencies were takenfrom the [1H,13C]-HSQC spectrum. Finally, the heavy-atomresonances of the asparagine and glutamine residues were assignedfrom the CBCA(CO)NH spectrum by connecting the 1H,15N side-chain amide resonances to the corresponding C!/C" or C"/C(chemical shifts while the side-chain NH2 were similarly obtained fromthe HBHA(CO)NH spectrum and checked using the 3D 15N-NOESY-HSQC spectra.

For the apo and HM-bound Y48A mutant, backbone amides wereassigned based on overlays and comparison of the [1H,15N]-HSQC

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spectra from the corresponding forms of the wt protein. Whenpossible, ambiguities were resolved by examining the 3D 15N-NOESY-HSQC spectra.Chemical shift perturbations of the 15N and 1H nuclei were analyzed

by overlaying the [1H,15N]-HSQC spectra of the apo and holoproteins and of the wt and Y48A mutant. The combined chemicalshifts perturbations (##avg) were derived from eq 1:

! ! !! = ! + ![( /6.51) ( ) ]avg N2

H2 0.5

(1)

where ##N and ##H are the chemical shift perturbations of the amidenitrogen and proton, respectively.NMR Backbone Dynamics Experiments. The backbone 15N R1,

R2, and Carr!Purcell!Meiboom!Gill (CPMG) R2 relaxationdispersion experiments were typically recorded at 600 MHz on "1.1mM [15N] single-labeled apo and HM-bound protein samples in 20mM sodium phosphate/100 mM NaCl pH 6.0/6% D2O. For the R1and R2 measurements, additional data were acquired at di"erentprotein concentrations and/or di"erent bu"ers (see main text fordetails). Relaxation values were obtained from series of 2Dexperiments with coherence selection achieved by pulse #eld gradientsusing the experiments described previously.31 The 15N R1 and R2relaxation rates were measured from spectra with di"erent relaxationdelays: 100 (in duplicate), 200, 300, 400, 500, 700, 900, 1200, and1500 (in duplicate) ms for R1, and 10 (in duplicate), 30 (in duplicate),50, 70, 90, 110, 150, and 190 ms for R2. The CPMG relaxationdispersion experiments32 were recorded with 0, 25, 50 (in duplicate),75, 125, 175, 225, 350, 550, 750 (in duplicate), and 1000 Hz pulserepetition rates. All data were processed in NMRPipe,26 and therelaxation parameters and their corresponding errors were extractedwith CCPN.27

NMR Relaxation Analysis. Starting from the experimental R2/R1values obtained for the 15N backbone atoms, the di"usion tensor of theapo FimH was obtained with R2R1_di"usion or Quadric_di"usionsoftware packages from A. Palmer’s lab. The residues with themeasured 15N R2 higher than one standard deviation from the average!R2" (obtained for the entire set of the FimH backbone amides) likelyexhibit signi#cant internal motions18 and, thus, were excluded from theanalysis. The model selection was performed using the F statistics asdescribed elsewhere.33

The R2/R1 and the hydrodynamic parameters for the FimHmonomer and dimer were calculated with HYDRONMR34 from theatomic coordinates of the apo FimH (lowest-energy NMR structure,this work) and the head-to-tail FimH!HM dimer (X-ray structure, thiswork), respectively. For the latter, the relaxation parameters wereaveraged over the values calculated for each of the two individualFimH molecules constituting the dimer. As explained above, theresidues with the measured 15N R2 higher than one standard deviationfrom !R2" were excluded from further analysis. For each backboneamide in the #nal data set, the population-weighted average of the R2/R1 ratios, (R2/R1)calcd,i, was obtained at varying populations of thedimer, p, eq 2:

= + "R R p R R p R R( / ) ( / ) (1 )( / )i i i2 1 calcd, 2 1 dimer, 2 1 monomer, (2)

At each p value, the agreement between the experimentally determined(R2/R1)exp and the (R2/R1)calcd for the entire protein was assessed bycalculating the Q factor (eq 3):

=# "

#Q

R R R R

R R

[( / ) ( / ) ]

( / )i i i

i i

2 1 calcd, 2 1 exp ,2

2 1 exp ,2

(3)

The lower the Q factor, the better the agreement between thecalculated parameters and the experimental data, with the best solutionfor the p value found at the global minimum of the Q = f(p) function(Figure 7B).NMR Di!usion Measurements. Samples containing "1.1 mM

[15N] single-labeled apo or HM-bound FimHL in 20 mM sodiumphosphate/100 mM NaCl pH 6.0/6% D2O were measured on theVarian NMR Direct-Drive Systems 800 MHz instrument. Dbppste-ghsqcse (with ni = 1 and phase = 1) experiments were run at 298 K

with a di"usion delay of 125 ms, and gradient amplitudes varied in therange of 6!60 G/cm (21 values arrayed). Signal attenuation was #ttedto the Stejskal!Tanner equation;35 the obtained average di"usionconstants (D) were D = 1.27 ± 0.01 ! 10!10 m2/s and D = 1.20 ± 0.01! 10!10 m2/s for the apo and HM-bound protein, respectively.

Solution Structure Calculation of the WT Apo FimHL. Theapo FimHL 3D

15N- and 13C-NOESY-HSQC spectra were peak pickedwith the assistance of the ATNOS software36 from the UNIO’10package (version 2.0.2). The extracted NOE cross-peaks were thenchecked manually prior to being used as an input for automatedassignment and structure calculations in CYANA version 2.113

together with a combination of dihedral restraints predicted fromTALOS+37 and DANGLE.38 Final structure re#nement in explicitsolvent was performed using the RECOORD protocol.39 The 25lowest-energy structures were retained for the #nal analysis andstructure validation in CING.14

Crystal Structure Determination of the WT FimHL!HM.Crystals of the HM-bound FimHL were observed after six months ofstorage at 277 K of a 0.5 mM sample ("8.5 mg/mL protein) in D2Ocontaining 20 mM sodium phosphate/100 mM NaCl at pH 6.0. Aseries of these crystals were cryoprotected by transferring them todrops consisting of 1 M HM in 20 mM sodium phosphate/100 mMNaCl pH 6.0/25% glycerol. The crystals were subsequently vitri#ed inliquid nitrogen. X-ray data were collected at the PROXIMA-1beamline of the SOLEIL synchrotron (Gif-Sur-Yvette, France). Thestructure was determined by molecular replacement using thestructure of apo-FimH (Protein Data Bank (PDB) number 4AUU)as a search model. The coordinates from heptyl !-D-mannose weredocked into the map from the initial molecular replacement modelresulting from PHASER, and the resulting model was subsequentlyre#ned. The #nal model was obtained after alternating cycles ofre#nement with phenix.ref ine40 and manual build using Coot41 and hasan Rfree of 15.5% and Rwork of 19.2% with excellent statistics (see TableS3, Supporting Information).

SAXS Studies of the WT Apo and HM-Bound FimHL. Smallangle X-ray scattering (SAXS) studies were conducted at the PETRA12 beamline (DESY, Hamburg, Germany). Prior to analysis, thesample was dialyzed into a bu"er matching the NMR conditions (20mM Mes, 100 mM NaCl pH 6.0) and concentrated (VivaspinHydrosart 5000 cuto") to 7.5 mg/mL (0.44 mM). The dialysis bu"erwas #ltered (0.22 %m) and kept as a bu"er blank for the experiments.For the apo FimHL, aliquots of the sample were prepared at di"erentconcentrations (2.0, 4.0, 6.0, and 7.5 mg/mL) by diluting the proteinstock with the #ltered dialysis bu"er. The holo FimHL stock wasprepared by adding a 5-fold excess of HM to the apo protein, anddi"erent concentrations of the holo FimHL were prepared as for theapo FimHL. For both forms, scattering curves were collected at thedi"erent protein concentrations and the bu"er was measured beforeand after each sample measurement. The samples were exposed for0.05 s to the beam with $ow of 0.2 mL/min. The collected SAXS datawere processed and analyzed with the ATSAS package.20

After bu"er subtraction, the scattering curves were compared. Forthe apo FimHL, this analysis revealed a slight concentration-dependency. The scattering curves were analyzed and mergedaccordingly to yield a #nal scattering curve,42 which was used for abinitio modeling. Brie$y, 20 ab initio models were generated byDAMMIF43 and averaged using DAMAVER.44 A starting bead modelwas extracted from the averaged dummy-atom model for thecalculation of a #nal model in DAMMIN.45 The scattering curve ofone of the structures of the NMR ensemble (model 21) was comparedto the experimental SAXS data set using CRYSOL.46

For the holo FimHL, the comparison of bu"er-subtracted scatteringcurves revealed a signi#cant concentration e"ect believed to be thee"ect of HM-induced FimHL dimerization. The scattering curvesmeasured at di"erent concentrations were not merged and were thustreated separately. Each curve was analyzed using OLIGOMER20 toobtain the fractions of monomeric and dimeric holo FimHL insolution.

The same SAXS measurements and data analysis were performedon apo and holo FimHL at pH 7.4. Therefore, samples at 2.0, 4.0, 6.0,

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and 8.2 mg/mL were prepared in the same way as above, except that20 mM HEPES, 150 mM NaCl pH 7.4 was used as dialysis anddilution bu"er.Isothermal Titration Calorimetry. Experiments were performed

at 25 °C in an ITC200 calorimeter (GE Healthcare, USA). All theprotein and sugar solutions were prepared in 20 mM sodiumphosphate/100 mM NaCl pH 6.0 and degassed before use. For thedirect titrations, the HM at 200 %M was titrated into a 10 %M solutionof either the FimHL wt or its Y48A mutant. For the reverse titration,the FimHL wt at 400 %M was titrated into HM at 12 %M. Titrationswere performed with 26 injections of 1.5 %L, using a delay betweeninjections of 140 s. In each case, the #rst data point was discarded, andthe integrated data were #tted to a binding model assuming n identicaland independent sites using MicroCal Origin 7.0.

! ASSOCIATED CONTENT*S Supporting InformationCING analysis of the apo FimHL NMR structure, RMSDbetween the apo FimHL NMR structure and deposited crystalstructures of FimHL complexes, details of the X-ray datacollection and re#nement of the FimHL!HM complex,[1H,15N]-HSQC spectrum of the apo wt FimHL, localRMSDs of the FimHL crystal structures, NOE coverage ofthe apo FimHL solution structure, backbone-amide NMRrelaxation pro#les of the apo and HM-bound wt and Y48AFimHL, SAXS scattering curves and data analysis of apo andHM-bound FimHL in HEPES bu"er, chemical shift perturba-tion analysis of Y48A FimHL, and the ITC titration of HM intoY48A FimHL. This material is available free of charge via theInternet at http://pubs.acs.org.Accession CodesThe FimHL solution structure and FimHL!HM crystalstructure have been deposited at the PDB data bank (entries3zpd and 4lov, respectively). All chemical shifts and T1 and T2relaxation data have been deposited at the BMRB (ID19066 forthe wt FimHL, ID19256 for the wt HM-bound FimHL,ID19255 for the apo Y48A FimHL mutant, and ID19254 forthe HM-bound Y48A FimHL mutant).

! AUTHOR INFORMATIONCorresponding Authors*Phone: (+32) 2 629 1025; fax: (+32) 2 629 1963; e-mail:ovolkov@vub.ac.be.*Phone: (+32) 2 629 3553; fax: (+32) 2 629 1963; e-mail:nvnuland@vub.ac.be.Present Address¶Unite ! de Glycobiologie Structurale et Fonctionnelle, UMR duCMRS 8576, Ba $timent C9, Avenue Mendeleiev, Universite ! desSciences et Technologies de Lille 1, 59 655 Villeneuve d’Ascq,France.NotesThe authors declare no competing #nancial interest.

! ACKNOWLEDGMENTSWe thank Klaartje Houben (Bijvoet Center for BiomolecularResearch, Utrecht University, The Netherlands), EvgenyTishchenko and Peter Sandor (Agilent, Santa Clara, CA) forthe acquisition of NMR data and useful discussions on theirinterpretation, and Tze Chieh Shiao (Department ofChemistry, Universite ! du Que !bec a " Montre !al, Canada) forthe !-D-heptyl mannose synthesis. Prof. De Greve (VUB,Brussels) is acknowledged for providing the template DNA,subsequently used in the PCR cloning of the expression vector

described in this work. We are grateful to the BIO-NMRTransnational Access program for providing the measurementtime at the Utrecht NMR facility (TA project BIO-NMR-00095). The Petra III (DESY, Hamburg, Germany) andPROXIMA1 (SOLEIL synchrotron Gif-sur-Yvette, France)beam lines are acknowledged for collection of the SAXS andcrystallographic data, respectively. S.V. acknowledges #nancialsupport of the framework for bilateral cooperation between theFonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO)and the Ministe "re du de !veloppement e !conomique, del’innovation et de l’exportation (MDEIE), Que !bec (researchproject G.A060.10N). The Hercules Foundation #nanced theequipment and infrastructure used in this work. A.N.V. andA.G.P. are FWO postdoctoral researchers, W.V. is a Brains Backto Brussels fellow, and Y.S. is a FWO predoctoral researcher.

! ABBREVIATIONS USEDAEBSF, 4-(2-aminoethyl)benzenesulfonyl $uoride hydrochlor-ide; DOSY, di"usion ordered spectroscopy; FimHL, FimHcarbohydrate-binding domain, also referred to as FimH lectindomain; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesul-fonic acid sodium salt; HM, !-D-heptyl mannose; heptyl !-D-mannopyranoside; IPTG, isopropyl "-D-1-thiogalactopyrano-side; ITC, isothermal titration calorimetry; Mes, 2-(N-morpholino)ethanesulfonic acid; UPEC, uropathogenic E.coli; upl, upper limit distances; UTI, urinary tract infection;SAXS, small angle X-ray scattering

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