Key Component of the Campylobacter Jejuni Iron Uptake System Scavenges Enterobactin Hydrolysis Product

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Bacteria in an intense competition for iron: Key component of the Campylobacter jejuni iron uptake system scavenges enterobactin hydrolysis product

Daniel J. Rainesa,OlgaV.Morozb, Elena V. Blagovab, Johan P. Turkenburgb, Keith S. Wilsonb,1, and Anne-K. Duhme-Klaira,1

aDepartment of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom; and bStructural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom

Edited by Kenneth N. Raymond, University of California, Berkeley, CA, and approved April 7, 2016 (received for review October 22, 2015) − To acquire essential Fe(III), bacteria produce and secrete siderophores [Fe(ENT)]3 is hydrolyzed by an intracellular esterase to release with high affinity and selectivity for Fe(III) to mediate its uptake into Fe(III) for use in the cell (8). the cell. Here, we show that the periplasmic binding protein CeuE of Along with the development of structurally diverse siderophores, Campylobacter jejuni, which was previously thought to bind the microorganisms adapted their associated receptor and transport

Fe(III) complex of the hexadentate siderophore enterobactin (Kd ∼ 0.4 ± proteins for the uptake of the appropriate Fe(III) complexes (9). To 0.1 μM), preferentially binds the Fe(III) complex of the tetradentate gain a competitive advantage, many bacteria have evolved to poach enterobactin hydrolysis product bis(2,3-dihydroxybenzoyl-L-Ser) siderophores produced by other bacteria. Campylobacter jejuni,for example, does not itself produce siderophores yet possesses an (H5-bisDHBS) (Kd = 10.1 ± 3.8 nM). The protein selects Λ-configured 2− uptake system that is able to use siderophores from competing [Fe(bisDHBS)] from a pool of diastereomeric Fe(III)-bisDHBS species 3− that includes complexes with metal-to-ligand ratios of 1:1 and 2:3. species (10). Initially, it was proposed that in C. jejuni [Fe(ENT)] is transported across the outer membrane by the receptors CfrA Cocrystal structures show that, in addition to electrostatic interactions 3− − and CfrB (11). Once in the periplasm, [Fe(ENT)] was proposed and hydrogen bonding, [Fe(bisDHBS)]2 binds through coordination to bind to the PBP CeuE, the resulting complex enabling the of His227 and Tyr288 to the iron center. Similar binding is observed transport of the ferric siderophore into the cytoplasm (12, 13). for the Fe(III) complex of the bidentate hydrolysis product 2,3- 3− In addition, increasing numbers of lower-denticity siderophores dihydroxybenzoyl-L-Ser, [Fe(monoDHBS)2] . The mutation of are being isolated from bacterial cultures and found to coordinate His227 and Tyr288 to noncoordinating residues (H227L/Y288F) resulted Fe(III) and mediate its uptake (14–18). For example, the trilactone 2− K ∼ ± μ in a substantial loss of affinity for [Fe(bisDHBS)] ( d 0.5 0.2 M). backbone of enterobactin makes it prone to hydrolysis, and al- These results suggest a previously unidentified role for CeuE within the though this lability is necessary to allow the intracellular release of Fe(III) uptake system of C. jejuni, provide a molecular-level understand- Fe(III) from the siderophore, it in addition leads to its slow deg- ing of the underlying binding pocket adaptations, and rationalize re- radation in aqueous media (7, 19–21). Three hydrolysis products C. jejuni Vibrio ports on the use of enterobactin hydrolysis products by , are formed: tris(2,3-dihydroxybenzoyl-L-Ser) (H7-trisDHBS), cholerae , and other bacteria with homologous periplasmic binding bis(2,3-dihydroxybenzoyl-L-Ser) (H5-bisDHBS), and 2,3-dihydroxy- proteins. benzoyl-L-Ser (H3-monoDHBS), with all three found in the growth medium of E. coli (Fig. 1). Although enterobactin, once secreted, is siderophore | Fe(III) uptake | periplasmic binding protein | enterobactin | also available to other cells (producers or nonproducers), it can Campylobacter jejuni Significance ith the rapid rise in bacterial resistance to antibiotics, a Wbetter understanding of cooperative behavior in microbial Almost all bacteria require Fe(III) for survival and growth. To communities is urgently needed for the development of novel ap- compete successfully for this essential nutrient, bacteria developed proaches to controlling infections caused by resistant bacteria (1, 2). very efficient Fe(III) uptake mechanisms based on high-affinity As an essential nutrient, iron is often a growth-limiting factor for Fe(III) chelators, so-called siderophores. To gain a competitive ad- beneficial, commensal, and pathogenic bacteria alike, not only vantage, many bacteria have evolved to scavenge and effectively due to its low solubility in water under aerobic conditions at and poach siderophores from other species. Enterobactin, one of the around neutral pH, but also because the host organism and com- strongest Fe(III) chelators known, is produced and secreted by peting microbes actively limit its availability (3, 4). Microorganisms many enteric bacteria. We show that a key protein involved in evolved efficient Fe(III) uptake mechanisms to overcome this Fe(III) uptake in the foodborne pathogen Campylobacter jejuni is challenge, a common strategy being the production of siderophores, adapted to scavenge enterobactin hydrolysis products, a strategy small Fe(III)-chelating molecules with high affinity and selectivity that may enable the pathogen to more efficiently exploit side- for Fe(III), with over 500 examples known to date (5). The sharing rophores produced by other bacteria and hence their resources. of siderophores is a recognized example for positive cooperativity that has been linked to bacterial virulence (6). The best-character- Author contributions: K.S.W. and A.-K.D.-K. designed research; D.J.R., O.V.M., E.V.B., and J.P.T. performed research; D.J.R., O.V.M., J.P.T., K.S.W., and A.-K.D.-K. analyzed data; and ized siderophores are hexadentate ligands that form coordinatively D.J.R., K.S.W., and A.-K.D.-K. wrote the paper. saturated, octahedral 1:1 complexes with Fe(III) (3, 5, 7), the most The authors declare no conflict of interest. studied being the triscatecholate enterobactin (H6-ENT) pro- This article is a PNAS Direct Submission. duced by many enteric bacteria (8). In Escherichia coli, enterobactin is synthesized within the cell Freely available online through the PNAS open access option. and secreted through the cell membranes to capture Fe(III) Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 5ADW and 5ADV). Experimental datasets from the environment. The resulting Fe(III)-enterobactin complex associated with this paper have been deposited with the University of York library (www. 3− [Fe(ENT)] is recognized by the outer membrane receptor FepA york.ac.uk/library/info-for/researchers/datasets/). and actively transported into the periplasm. In the periplasm, 1To whom correspondence may be addressed. Email: [email protected] or 3− [Fe(ENT)] is sequestered by the periplasmic binding protein [email protected]. (PBP) FepB, which transfers it to an inner membrane trans- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. porter for further transport into the cytoplasm (9). Once there, 1073/pnas.1520829113/-/DCSupplemental.

5850–5855 | PNAS | May 24, 2016 | vol. 113 | no. 21 www.pnas.org/cgi/doi/10.1073/pnas.1520829113 Downloaded by guest on September 30, 2021 Fig. 1. Molecular structure of enterobactin, its hy-

drolysis products, the siderophore mimic H6-MECAM, and a selection of tetradentate siderophores.

only be used once because Fe(III) release requires its hydrolysis. synthesize H -bisDHBS, with modifications aimed at simplifying the 5 − The enterobactin hydrolysis products, however, could be used again procedures (28). The Fe(III) complex of bisDHBS5 was soaked as secondary, lower-denticity siderophores. into apo-CeuE crystals, grown as reported previously (27). Experi- It has been demonstrated that the human pathogens C. jejuni mental details for the preparation of the Fe(III) complex of − (10, 22, 23) and Vibrio cholerae (24), the causes of food poisoning bisDHBS5 , crystal growth conditions, data collection, and re- and cholera, respectively, can use enterobactin hydrolysis products finement are provided in Supporting Information. Crystallographic for the uptake of Fe(III) from their environment. Both are known statistics and a discussion of unit cell dimensions and average B not to produce enterobactin. values can be found in Supporting Information (Table S1). An alternative Campylobacter Fe(III) acquisition model that relies Three crystals were selected; crystal I was soaked for 90 min, on these linear hydrolysis products was recently proposed based on crystal II for 24 h (PDB ID code 5ADW), and crystal III for 11 d thefindingthatCee,thesoletrilactoneesteraseofC. jejuni and (PDB ID code 5ADV). As expected, all three crystals were in space Campylobacter coli, is located in the periplasm, i.e., these bacteria are group P1 with three protein monomers per asymmetric unit, con- unable to degrade enterobactin within the cytoplasm (25). The model sistent with the apo-CeuE structure (PDB ID code 3ZKW) (27). CHEMISTRY suggests that, once the Fe(III) complex of enterobactin enters the Crystal I showed no additional electron density in the CeuE binding periplasm, its ester backbone is cleaved by Cee, which is highly ef- pockets, indicating that the 90-min soaking time was too short. ficient in hydrolyzing both the Fe(III) complex and apo-enterobactin. Crystal II has additional electron density in the binding pockets of each of the three independent CeuE monomers, which was The resulting hydrolysis products, mainly the tetradentate ligand 2− 5− 3− modeled as [Fe(bisDHBS)] inchainsAandB(Fig.2andFig. bisDHBS and the bidentate ligand monoDHBS , are then used to − S1). CeuE binds a single [Fe(bisDHBS)]2 species per monomer in mediate the subsequent transport of Fe(III) into the cytoplasm. 5− The identification of the esterase Cee and in particular the ob- an Fe(III):siderophore:protein ratio of 1:1:1. BisDHBS chelates − servation that bisDHBS5 can be used independently of enter- the Fe(III) center in a tetradentate fashion with the four cat-

obactin (25) raise important questions about the siderophore echolate oxygen donors coordinated. The remaining two co- BIOCHEMISTRY preference of the PBP CeuE and its role in the iron uptake in ordination sites of the distorted octahedral Fe(III) center are C. jejuni. By using siderophore mimics, we previously demonstrated occupied by donor atoms provided by the side chains of His227 and that CeuE can bind the Fe(III) complexes of both hexadentate and Tyr288. In addition, electrostatic interactions and hydrogen bonds involving residues Arg118, Arg205, Arg249, and Lys121 contribute tetradentate catecholate ligands (26, 27). Our cocrystal structures − to the binding (Fig. 3). The diserine linker in bisDHBS5 forces the revealed that CeuE interacts with the coordinatively saturated − oxygen donors in the ortho and meta position to the amide groups Fe(III) complex of the hexadentate mimic MECAM6 through on the catechol units to occupy adjacent coordination sites on the electrostatic interactions and hydrogen bonding, whereas it binds Fe(III) center. Consequently, the remaining two coordination sites the coordinatively unsaturated complex of the tetradentate mimic 4− are not equivalent. The site opposite the weaker catecholate donor 4-LICAM by recruiting the side chains of two amino acid resi- (ortho position) is occupied by the oxygen donor of Tyr288, dues (His227 and Tyr288) to complete the coordination sphere of whereas that opposite the stronger oxygen donor (meta position) is the Fe(III) center. We established that His227 and Tyr288 are occupied by the nitrogen donor of His227, consistent with their conserved among a subset of related PBPs, including VctP from respective trans-influence (29). Although the observed Fe(III) V. cholerae, and suggested that this subset of PBPs undergo similar binding mode is similar to that which we reported previously for − structural changes to adapt to the binding of lower-denticity sidero- the C2-symmetric siderophore mimic 4-LICAM4 (PDB ID code phores (27). The recent report that V. cholerae most efficiently uses 5− 7− 5− 5A1J)(27),thediserinebackboneofbisDHBS plays a key role in trisDHBS and bisDHBS for the acquisition of Fe(III) provided determining the orientation of the unsymmetrical natural sidero- a partial confirmation of this prediction (24). phore. In both chains A and B, the carboxylate group in the 2− − Here, we report that CeuE binds [Fe(bisDHBS)] with much backbone of bisDHBS5 provides an additional negative charge higher affinity than the Fe(III) complex of enterobactin, reveal the and accepts a hydrogen bond from Arg249 (Fig. 3). This high structural basis for this difference in binding strength, and examine degree of complementarity indicates that the binding pocket of key aspects of the relevant Fe(III) coordination chemistry in solution. CeuE is perfectly suited to the binding of the tetradentate ligand. In the binding pocket of chain C, there was much less density and Results it was modeled as a half-occupied Fe(III) center with no co- 2− 3− Crystal Structures of CeuE-[Fe(bisDHBS)] and CeuE-[Fe(monoDHBS)2] . ordinating ligand. The anomalous difference Fourier maps confirm A previously reported total synthesis of enterobactin was adapted to a peak of about 50% of that seen for the Fe(III) in chains A and B.

Raines et al. PNAS | May 24, 2016 | vol. 113 | no. 21 | 5851 Downloaded by guest on September 30, 2021 structures prompted us to investigate whether this complex is also the predominant species formed in solution. Due to the mismatch between the four donor atoms provided by the two chelating catechol − units of bisDHBS5 and the preferred sixfold coordination environment of Fe(III), the composition and structure of the complex(es) formed have long been a matter of debate. Based on a UV-visible absorbance spectroscopic study carried out in aqueous solution at − pH 9, it was suggested that bisDHBS5 forms a 1:1 complex with Fe(III) under these conditions and that the remaining two coordination sites are occupied by solvent molecules (15). In contrast, NMR spectroscopic investigations carried out in d6-DMSO with Ga(III) as a diamagnetic substitute for Fe(III) indicated a metal- to-ligand ratio of 2:3. Consequently, the formation of a dinuclear complex of composition {Fe2(bisDHBS)3} was proposed (32). Stoichiometric control allowed the isolation of two different Fe(III) complexes of a tetradentate siderophore mimic that con- sists of two ethyl-bridged catecholamides. A metal-to-ligand ratio of 2:3 resulted in the crystallization of a dinuclear triple-stranded 6− helicate of composition [Fe2(L)3] , whereas a basic solution with metal-to-ligand ratio of 1:1 produced crystals of hydroxo-bridged 4− dinuclear complexes of composition [{Fe(L)(OH)}2] (29). We carried out experiments to rationalize these observations and related the results to biologically relevant conditions. Fig. 2. (A) Ribbon representation of the three independent CeuE chains in the To probe the metal-to-ligand ratio, we used Job’smethodof unit cell of crystal II (PDB ID code 5ADW) with a black dashed line representing continuous variation (33, 34), monitoring complex formation by steric clash of chain B N terminus with the binding pocket of chain C. (B–D)Cylinder both electronic absorbance and CD spectroscopies. The spectro- representation of the different ligand binding orientations found in crystals II and scopic measurements were carried out in aqueous solution [5% σ III with electron density shown as 2Fobs-Fcalc, contoured at 1 :(B)crystalIIchain (vol/vol) DMSO] at pH 7.5 using Fe(III) nitrate as the Fe(III) B; (C) crystal III chain A; and (D) crystal III chain C. Ligands are shown as cylinders: source. Nitrilotriacetic acid (NTA) was added to prevent the pre- gray, carbon; blue, nitrogen; red, oxygen; and iron, coral; and Arg, Tyr, and His side cipitation of Fe(III) hydroxides. Control experiments ascertained chains are shown as cylinders with carbon atoms colored as their ribbons. that the presence of NTA does not interfere with the reaction of − Fe(III) with bisDHBS5 under these conditions. Electronic absorbance spectra were recorded at selected metal- There is some residual density around the Fe(III), but it was not − − to-ligand ratios in the region of the ligand-to-metal charge transfer 5 3 − possible to satisfactorily model this as bisDHBS or monoDHBS . (LMCT) band of [Fe(bisDHBS)]2 (Fig. 4A). The maximum of the Although the binding pockets of chains A and B are both open to LMCT band shifts significantly upon variation of the metal-to- solvent, access to the pocket of chain C is restricted by the N ter- ligand ratio. A red complex forms under conditions of excess li- minus of chain B. The resulting steric constraints explain the only gand. The λmax at ∼510 nm is consistent with the formation of a partial occupation of this pocket (Fig. 2A). tris(catecholate) species (35, 36). When the metal-to-ligand ratio Crystal III has similar cell dimensions and packing to crystal II approaches 1:1, the color of the solution changes from red to purple. (Table S2). There is electron density in each of the three binding The observed λ of around 565 nm is consistent with the for- pockets (Figs. S1 and S2), the density is weak in the region corre- max − mation of a bis(catecholate) species (36, 37). The Job plot confirms sponding to the backbone of bisDHBS5 (below the 1σ level), sug- − that a λ around 510 nm is consistent with a metal-to-ligand ratio gesting that the di-serine ester-linkage of bisDHBS5 was highly max of 2:3, whereas a λmax around 565 nm is consistent with a metal-to- mobile or had hydrolyzed or a combination of both. The best fit ligand ratio of 1:1 (Fig. 4B). to the density was attained when chain B was modeled with − In summary, the electronic absorbance spectra demonstrate that [Fe(bisDHBS)]2 bound while chains A and C were modeled with − in aqueous solution near neutral pH, both 1:1 and 2:3 complexes two monoDHBS3 ligands coordinating the Fe(III) center (Fig. 2). 3− are formed upon reaction of Fe(III) with H5-bisDHBS (Fig. S4), Remarkably, the orientation of the monoDHBS ligands in chain C with the species distribution being controlled by stoichiometry. differed from that in chain A. In A, the oxygen donors in both the Similarly, the visible region CD spectra vary with the metal-to- ortho and meta position of the catecholamide units occupy adjacent − ligand ratio (Fig. 5A). At metal-to-ligand ratios of 3:2 and 1:1, the coordination sites on the Fe(III) center, as seen in [Fe(bisDHBS)]2 . − In contrast, in chain C, one of the monoDHBS3 ligands was in a flipped position, with the attached serine of the backbone pointing into the binding pocket. Here, the donors in the ortho position of the two catecholamide units coordinate trans to one another, whereas the donors in the meta position remain coordinated cis to one another. In this isomer, His227 and Tyr288 occupy coordination sites trans to the catecholate oxygen donor atoms in the meta position (Fig. 2). This change in orientation of the ligands is likely to be due to steric constraints imposed by the N terminus of chain B (Fig. S3). Crystal III thus reveals two alternative binding modes for the Fe(III) complex − of the monomeric enterobactin hydrolysis product monoDHBS3 . In crystals II and III, the Fe(III) complexes are bound with the metal center in the Λ-configuration, as previously observed in the 6− cocrystal structures of (CeuE)2-[{Fe(MECAM)}2] (26), CeuE- − − 3 − [Fe(4-LICAM)] (27), FeuA-[Fe(bacillibactin)] (30), FeuA- Fig. 3. (A) Overlay of the three independent [Fe(bisDHBS)]2 units and key residues 3− 3− [Fe(MECAM)] , and FeuA-[Fe(ENT)] (31). in the respective substrate binding pocket of CeuE shown as cylinders with the carbon atoms colored according to chain: crystal II chains A (blue) and B (gold), crystal Relevant Fe(III) Coordination Chemistry in Solution. The identification III chain B (green), dashed H bonds exemplified for crystal II chain B. (B) Schematic 2− of the Λ-configured 1:1 complex [Fe(bisDHBS)] in the cocrystal diagram of key hydrogen-bonding interactions (charges omitted for clarity).

5852 | www.pnas.org/cgi/doi/10.1073/pnas.1520829113 Raines et al. Downloaded by guest on September 30, 2021 Similar observations were reported for the amonabactins, a family of bis(catecholate) siderophores in which the catecholate units are at- e tached to the N atom of lysine residues present in their peptide-based backbones(16,42,43).However,the increase in Cotton effect upon decrease of the metal-to-ligand ratio from 1:1 to 2:3 is more pronounced with H5-bisDHBS than for the amonabactins. This is probably α due to a stronger chiral induction from the C of the serine backbone α in the linear dimer, which is closer to the Fe(III) center than the C of the lysine residues in the backbone of the amonabactins (Fig. 1).

Interactions Between CeuE and the Fe(III) Complexes of Siderophores in Solution. CD. To probe the stereochemistry of the Fe(III) center in the presence of CeuE in solution, CD spectra were recorded in the

Fig. 4. (A) Selected electronic absorbance spectra [colors from black to light blue ordered by metal-to-ligand ratio as indicated; [M] + [L] = 0.4mM;0.1M CHEMISTRY Tris·HCl with 5% (vol/vol) DMSO; pH 7.5]. (B) Job plot for the reaction of

H5-bisDHBS with Fe(III), obtained by following the absorbance at 512 nm (red triangles) and 563 nm (blue circles). The absorbance values are averages of two experiments (error bars indicate the difference between the runs).

CD spectra show an intense positive band around 320 nm, a weaker positive band around 410 nm, and a weak bisignate band centered at 595 nm, which is near the LMCT absorption maximum of the 1:1 BIOCHEMISTRY species. Scarrow et al. (15) reported a similar CD spectrum for − [Fe(bisDHBS)]2 isolated from growth cultures and assigned the intense near-UV band to ligand-based transitions and the weaker visible region features to LMCT transitions. Because the latter are indicative of the chirality at the Fe(III) center, the low intensity of the signal indicates that the chiral induction by the L-serine residues in the backbone is only weak. Upon changing the metal-to-ligand ratio to 2:3, the maximum of the near-UV band shifts from 320 to 340 nm and the visible region now shows a bisignate band with a positive signal at 415 nm and a negative signal at 550 nm, consistent with a negative Cotton effect. The position of the zero CD crossover at 503 nm is close to the − LMCT absorption maximum (∼510 nm) of the Fe(III)-bisDHBS5 2:3 species. The resulting CD spectrum is characteristic of a solution of Fe(III) tris(catecholamide) complexes in which the Δ-configuration dominates (38). This characteristic change in the CD profile coincides with the color change from purple to red reflected in the electronic absorbance spectra (Fig. 4A). The concomitant increase in amplitude is consistent with the increase in the number of coordinated catechol units, not only because each is expected to contribute to the chiral induction effect, but also due to the pairwise

additivity of exciton-coupled chromophores (39). The effect is also Fig. 5. (A) CD spectra of solutions of H5-bisDHBS or Fe(III) plus H5-bisDHBS at consistent with the diastereoselective formation of a dinuclear triple key metal-to-ligand ratios [0.1 M Tris·HCl buffer with 5% (vol/vol) DMSO, pH 7.5, helicate, in which the enantiomerically pure ligand predetermines 150 mM NaCl, [M] + [L] = 0.4 mM]. (B and C) CD spectra of solutions of Fe(III) plus

the helical direction and hence the chirality of both metal centers, H5-bisDHBS (B) or Fe(III) plus enterobactin (C) recorded after addition of increasing as seen in similar supramolecular assemblies (40, 41). increments of CeuE as indicated (0.1 M Tris·HCl buffer, pH 7.5, 150 mM NaCl).

Raines et al. PNAS | May 24, 2016 | vol. 113 | no. 21 | 5853 Downloaded by guest on September 30, 2021 wavelength range of the LMCT band. Series of spectra were Discussion obtained during a titration of a solution of CeuE into solutions of Enterobactin is one of the most efficient mediators of bacterial Fe(III) plus H5-bisDHBS or Fe(III) plus enterobactin in buffer (pH Fe(III) uptake, and increasing evidence indicates that its hydro- − 7.5). The titrations allowed the changes in spectral features upon lysis products, in particular bisDHBS5 , play an important role in binding of the respective Fe(III) siderophore complex to CeuE to the competition for essential Fe(III) because they can be exploited be observed (Fig. 5 B and C). by siderophore poachers, such as C. jejuni. Our studies demonstrate that CeuE, the periplasmic binding The first CD spectrum was obtained after addition of Fe(III) and − protein of C. jejuni,selectsΛ-[Fe(bisDHBS)]2 from a mixture of H5-bisDHBS to the buffer, followed by equilibration. It confirms − 5− 5 that, in the absence of CeuE, the L-serine backbone in bisDHBS species in solution. In the absence of the protein, bisDHBS was has only a weak influence on the stereochemistry at the Fe(III) found to coordinate to Fe(III) in metal-to-ligand ratios of both 1:1 center, with Δ-configured complexes in slight excess. and 2:3, depending on the relative concentrations of Fe(III) and siderophore present in solution. In both complexes, chiral in- The spectra recorded upon addition of CeuE indicate inversion of α duction from the C of the L-serine backbone causes the Fe(III) configuration at the metal center with isodichroic points at 305 and Δ 544 nm. The positive band at 320 nm changes into a negative band at center(s) to adopt preferentially the -configuration in aqueous 330 nm. The weak positive signal across the LMCT range develops buffer at pH 7.4, with weak (1:1 complex) to moderate (2:3 complex) diastereoselectivity. The ability of CeuE to select a defined into a broad negative band with a minimum at 395 nm and a positive Λ Fe(III) complex from a mixture is consistent with its role in the band with a maximum at 600 nm, consistent with a -configured Fe(III) transport process, where it functions as a periplasmic Fe(III) center (38). The series of spectra confirms that CeuE selects Λ– 2− gatekeeper and is required to deliver the correct Fe(III) complex to for configured [Fe(bisDHBS)] and shows that the diastereo- the inner membrane transporter. CD spectra recorded during the − isomers that remain in solution are able to reequilibrate quickly. titration of CeuE into a solution of [Fe(bisDBHS)]2 indicated an For comparison, an analogous titration was carried out with a inversion of configuration from Δ to Λ upon addition of the pro- solution containing Fe(III) and enterobactin (Fig. 5C). As expected 3− tein. The effect is caused by CeuE driving the equilibrium by from previous reports, the initial CD spectrum of [Fe(ENT)] selecting for Λ-configured complexes; the two diastereomers that is characteristic of Fe(III) tris(catecholamide) complexes with remain in solution reequilibrate quickly. Δ-configuration (38). Upon addition of CeuE, the bands decrease Of particular significance is the revelation that CeuE binds − − in intensity, indicating a shift in the equilibrium between Δ-and [Fe(bisDBHS)]2 ∼100 times more tightly than [Fe(ENT)]3 ,with Λ-configured complexes is caused by the Fe(III) complex of K = 10.1 ± 3.8 nM and K ∼ 0.4 ± 0.1 μM, respectively. This marked d d − enterobactin binding to CeuE. However, even after addition of 1.6 difference strongly suggests that [Fe(bisDHBS)]2 is the preferred − − equivalents of CeuE, Δ-configured [Fe(ENT)]3 still predominates. ligand for the protein rather than [Fe(ENT)]3 as previously thought. The observed selection of Λ-configured complexes by CeuE indi- Our cocrystal structures provide molecular-level insights into the cates that the chiral L-serine backbone, which has a Δ-configuration– binding pocket adaptations that give rise to this pronounced dif- inducing effect, slightly hinders rather than facilitates protein binding. ference in affinity and are the first (to our knowledge) to reveal the The resulting moderation of binding affinity is consistent with the interactions of a natural tetradentate siderophore with its cognate PBP. In addition to electrostatic and hydrogen-bonding interac- intermediary role of the periplasmic binding protein within the iron 2− transport pathway. A precise tuning of affinity is essential to allow the tions between the binding pocket and [Fe(bisDHBS)] , the crystal transfer of the cargo to the inner membrane transporter in the final structure showed the direct coordination of two amino acid side chains (His227 and Tyr288) to the Fe(III) center. In contrast, the step of the Fe(III) transport chain. binding of the Fe(III) complex of MECAM, the hexadentate Fluorescence spectroscopy. To allow a quantitative comparison of the − enterobactin mimic, was found earlier to rely solely on electrostatic binding strength, the affinity of CeuE for [Fe(bisDHBS)]2 and − interactions and hydrogen bonding (26). The oxygen donor of 3 − [Fe(ENT)] was determined using intrinsic fluorescence quenching Tyr288, which is directly coordinated in CeuE-[Fe(bisDHBS)]2 , experiments. Aliquots of the respective Fe(III)-siderophore solution is positioned 3.5 Å away from the metal center in (CeuE)2- (1:1 ratio, 12 μMinaqueous40mMTris·HCl, 150 mM NaCl) were 6− [{Fe(MECAM)}2] , where it participates in hydrogen bonding. added successively to a solution of CeuE (240 nM in aqueous 40 mM His227 is disordered forming part of a flexible loop in both the − Tris·HCl, 150 mM NaCl, pH 7.5) and the fluorescence intensity was apo-CeuE and (CeuE) -[{Fe(MECAM)} ]6 structures. Hence, we λ = λ = – 2 2 measured after equilibration ( exc 280, em 310 410 nm) (Fig. propose that His227 and Tyr288 enable CeuE to preferentially bind S5). The dissociation constants (Kd) were obtained from three in- the tetradentate enterobactin hydrolysis product rather than intact dependent measurements, as described in Supporting Information. hexadentate enterobactin. This proposal is supported by the observa- − Surprisingly, CeuE was found to bind [Fe(bisDHBS)]2 tion that the replacement of His227 and Tyr288 with noncoordinating 3− 2− with much higher affinity (Kd = 10.1 ± 3.8 nM) than [Fe(ENT)] amino acids leads to a drastic drop in affinity for [Fe(bisDHBS)] . (Kd ∼ 0.4 ± 0.1 μM). This observation shows that the direct co- There is an equivalent direct coordination of His227 and Tyr288 3− ordination of the His227 and Tyr288 side chains to the Fe(III) in the crystal structure of CeuE-[Fe(monoDHBS)2] ,inwhichtwo 3− 5− center more than compensates for the loss of electrostatic attraction monoDHBS ligands, the hydrolysis products of bisDHBS ,co- caused by the decrease in negative charge upon moving from a ordinate to the Fe(III) center. In both crystals, the Fe(III) com- Λ hexadentate to a tetradentate catecholate siderophore. plexes are bound with the metal center in -configuration. To confirm the importance of His227 and Tyr288 for the binding A sequence homology search of reported 3D structures (Fig. − affinity of CeuE to [Fe(bisDHBS)]2 , site-directed mutagenesis was S6) suggests that a subset of related Fe(III)-siderophore binding used to replace the Fe(III)-coordinating amino acids with non- proteins shares a preference for the hydrolysis products of enter- coordinating, yet sterically similar residues. As anticipated, mutation obactin over intact enterobactin. The PBP of the Vct uptake system in V. cholerae has a high similarity to CeuE and contains the co- of His227 to leucine (H227L) and Tyr288 to phenylalanine (Y288F) ordinating histidine and tyrosine residues (27). Hence, our results resulted in a clear drop in binding affinity. The dissociation constant rationalize the recent discovery that V. cholerae is able to use the obtained with the double mutant (Kd ∼ 0.5 ± 0.2 μM) indicates 3− linear derivatives of enterobactin, but not enterobactin itself (24). weak binding, comparable to that observed for CeuE-[Fe(ENT)] We therefore propose that CeuE preferentially selects for − (Kd ∼ 0.4 ± 0.1 μM). Both dissociation constants are about two 2 2− [Fe(bisDHBS)] in the periplasm and mediates its uptake into the orders of magnitude higher than that for CeuE-[Fe(bisDHBS)] cell. As a result, the Fe(III) complex of enterobactin is retarded in (Kd = 10.1 ± 3.8 nM). In contrast, the dissociation constant obtained − the periplasm where it is exposed to esterase-catalyzed hydrolysis to for CeuE-[Fe(bisDHBS)]2 is similar to those previously published its linear components. Because it has been reported that C. jejuni − for the binding of hexadentate tris(catecholate) siderophore com- can use bisDHBS5 as a siderophore independently of enterobactin plexes to PBPs, such as FeuA and FepB (10–50 nM) (30, 31, 44–46). (10) and the discovery of the trilactone esterase Cee in the periplasm

5854 | www.pnas.org/cgi/doi/10.1073/pnas.1520829113 Raines et al. Downloaded by guest on September 30, 2021 of C. jejuni (25), it appears that CeuE, previously considered to be a the uptake of Fe(III) by Campylobacter and a number of other − [Fe(ENT)]3 binding protein, is instead adapted to preferentially − pathogenic bacteria. bind [Fe(bisDHBS)]2 . This ability to use the enterobactin hydrolysis product for Fe(III) uptake and hence to avoid the metabolic costs of Methods siderophore production provides C. jejuni with a competitive ad- Experimental details including general methods, compound synthesis (Fig. S7)and vantage when it encounters enterobactin-secreting bacteria either characterization, site-directed mutagenesis, protein crystallization, X-ray data within the host or contaminated water. collection, structure solution and refinement (Tables S1 and S2), and fluorescence In contrast, in E. coli, the PBP FepB does not possess equivalent quenching data (Figs. S5 and S8)canbefoundinSupporting Information. tyrosine and hisitidine residues and the esterase that cleaves the Fe(III) complex of enterobactin is located in the cytoplasm. Con- ACKNOWLEDGMENTS. We thank Mr. S. Hart for assistance with data sequently, the Fe(III)-siderophore uptake systems of the side- collection, Dr. A. Leech for help with CD spectroscopy, and the Diamond rophore scavenger C. jejuni and the siderophore producer E. coli Light Source for access to beamlines I02 and I04 (proposal; mx-7864). We show significant differences. thank the UK Biotechnology and Biological Sciences Research Council and Taken together, these insights strongly suggest an important the Engineering and Physical Sciences Research Council (Grant EP/L024829/1) in vivo role for the tetradentate siderophore and its cognate PBP in for financial support.

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