Structural biology

The structure of PfeA has been determined to 2.1 Å resolution with residues 15–721 of PfeA fitted into experimental density. PfeA has the same two domains (22-stranded β-barrel and N-terminal plug) common to the TBDTs (Fig. 1b). Extracellular loops of the plug domain and the β-barrel are denoted NL1 to NL3 and L1 to L11, respectively (Supplementary Fig. 1). The periplasmic turns connecting to β strands are denoted T1 to T10. PfeA has a 75% sequence homology (60% identity) with E. coli TBDT FepA (1FEP), which is also its closest structural relative with a root-mean-square deviation (r.m.s.d.) of 1.01 Å for 652 aligned Cα atoms. The P. aeruginosa TBDT, PaPirA (5FP2, 72% homology and 60% identity) and the Acinetobacter baumannii homologue AbPirA (5FR8, 64% homology and 48% identity)31, both involved in catechol–siderophore uptake, superpose with a r.m.s.d. of 1.15 Å for 480 residues and 1.06 Å for 656 residues, respectively. These sequence and structural similarities are mirrored by the observation that all these TBDTs have been shown to transport ferric-enterobactin32. PfeA has loops (L3, L4, L7 and L10) that cover the extracellular face of the structure and limit the access to the plug domain compared with the other TBDTs (Supplementary Fig. 2a). A similar arrangement is seen in the FepA crystal structure, but the loops are not fully ordered in that structure. A space-filling analysis of this extracellular face reveals only a narrow tunnel with an approximate diameter of 3 Å (NE2 Gln482 to OE1 Glu327 and Asn268 ND2 to Ser479 OG). The tunnel leads to a polar void deeper inside the structure that is formed by conserved residues Gln67 of NL1, residues 100–106 of NL3, Gln267 and Asn268 of L3, residues 321–322 and Glu327 of L4, residues 477–479 and 482–483 of L7 and Tyr641. A molecule of ethylene glycol is located here forming hydrogen bonds with the side chain of Arg100 and backbone nitrogen of Trp103 (Supplementary Fig. 3a). There is a further smaller void deeper in the pore formed by Arg68, 98–102, 106–108, Asn315, 317–318, 320–321, Gly328, Tyr338, Ala340, Asp376, Ser378.

Co-crystallisation efforts were unsuccessful possibly due to instability of the Fe3+-enterobactin, which underwent colour changes and precipitated during co-crystallisation experiments. Soaking of native crystals with Fe3+-enterobactin prior to data collection resulted in colour changes in the crystals. The data collected from such crystals revealed additional difference electron density that could unambiguously be modelled as a single Fe3+-enterobactin molecule (Fig. 2a). The ferric-siderophore is bound to PfeA by residues from loops L2, L3, L4, L7 and L11 (Fig. 2b) on the extracellular surface, and is partly exposed to solvent. The molecule is not bound inside the cavities we identified in the apo-structure rather it is located in the entrance of the polar cavity that binds ethylene glycol. For ease of discussion, we split the enterobactin molecule into three catechol rings that we number I, II and III (Fig. 2c). The side chain of Arg480 sits between rings I and II and makes electrostatic/cation-π interactions with ring II. The side-chain of Gln482 sits between the catechol rings I and III whilst loop L4 sits between rings II and III. The catechol ring III is buried from solvent by the protein, the other two rings are partially exposed to solvent (Supplementary Fig. 2b, c). Each ring makes specific hydrogen bonds to the protein (Fig. 2c; Supplementary Fig. 4a). The oxygen atoms O6 and O3 of catecholate III hydrogen bond to Ser479 and Gln482; O5 and O2 of the catecholate II hydrogen bond to the main chain nitrogen atoms of Arg480 and Gly325, whilst O4 of catecholate I hydrogen bonds to backbone nitrogen atom of Gln482. In addition, both Gln482 and Gln219 make hydrogen bonds with the ester of the trilactone backbone. As well as hydrogen bonds, there are extensive van der Waals contacts involving asparagine residues 268 and 270, glycines 481 and 483, valine residues 695 and 696 and Ala323, Gly324, Thr326 of loop L4 within the binding pocket. There are no π–π stacking interactions in this binding site. The more confidently identified of the two iron atoms (using anomalous diffraction) in the FepA structure was located in the vicinity of Lys483 (Arg480 in PfeA) and therefore occupies a similar position to that observed in our structure. Lys483 of FepA was also confirmed to be involved in the binding by mutation studies33. The second iron site in the FepA structure was identified as close to the first iron, but it was not described in detail14. Superposition of PfeA-Fe3+-enterobactin structure with the previously reported FhuA-Fe3+-ferrichrome (and related TBDT structures) show that these other transporters bind their cognate siderophores in a different location than observed here in PfeA (Supplementary Fig. 5).

Fig. 2 Complex structure of PfeA with Fe3+- enterobactin. a F O –F C electron density omit map at 3 σ around Fe3+-enterobactin complex. Enterobactin is shown as sticks with carbon atoms coloured in blue, nitrogen in dark blue and oxygen in red. The Fe3+ is represented as an orange sphere. b Fe3+-enterobactin binds to the extracellular loops. The N-terminal plug domain is coloured in blue, the β-barrel in yellow and the NL1 and NL3 loops in orange. Secondary structure elements involved in the binding site have been labelled. c Binding site of the siderophore. Residues within 4.0 Å of the siderophore are displayed and hydrogen bonds are shown as black broken lines. d Comparison of the apo (green) and complex (yellow) structures. Loops NL1, L4, and L11 which undergo a large change of conformation have been highlighted in magenta Full size image

The polar void described in the apo-structure is also seen in the Fe3+-enterobactin complex structure. This void is connected to the enterobactin binding site and is formed by Gln67 of NL3, Trp103, Arg104 of NL3, Gln267, Asn268, Glu327, Leu477, Tyr641. Comparison of the Fe3+-enterobactin complex structure with the apo-structure shows that L4 has undergone a large change (6.3 Å shift between Cα of G325) of conformation in order to interact with enterobactin molecule (Fig. 2d). This loop contains multiple glycines (321GLAGGTEG328) that are well known to confer structural flexibility. Three of these glycines are conserved in FepA, AbPirA and PaPirA (G321, G324 and G328). The change in conformation of Glu327 of L4 results in the side-chain reaching further inside the pore to make a salt bridge with Arg100 (Fig. 3a). Since the size and shape of the polar void changes upon ligand binding due to the movement of Glu327, we term this region the ‘gateway'. Ring III of the enterobactin points into the ‘gateway’. The gateway is connected to what we now term the internal cavity (similar to the second smaller void in the apo-structure) which is formed by Arg68, residues 99–102, 106–109, Gln267, Glu320, Leu322, 327–328, Tyr338, 378–379. In the other transporters, the cognate ligand is bound at the interface of the gateway and the internal cavity.

Fig. 3 Conformation changes induced by the Fe3+-enterobactin binding. Apo and complex structures are represented in green and yellow, respectively. a In the extracellular side, the movement of the loop L4 allows Glu327 to interact with Arg100 and push away Glu106 of NL3 that displaces loop NL1. b In the periplasmic side, the N-terminal part of PfeA rearranges following the Fe3+-enterobactin binding. The peptide bond of the Pro28 flips resulting in the formation of β-sheet rather the α-helix observed in the apo-structure. In consequence, the TonB box become disordered. T24* refers to the threonine 24 of the complex structure Full size image

Concomitant with the formation of the Glu327-Arg100 salt bridge, Glu106 of NL3 (which in the apo-structure is salt-bridged to Arg100) adjusts its position. The shift of Glu106 is accompanied by a movement of 3.1 Å of NL1 of the plug domain centred on Ser63 (Fig. 3a). We also observe conformational changes in the N-terminal portion of the protein (Fig. 3b). In the apo-structure, residues 20–26 form an α-helix, and the TonB box motif (13EQTVVATAQ21) that engages TonB is mostly well ordered. In the Fe3+-enterobactin complex, the peptide bond at Pro28 (β-turn type II) flips resulting in the formation of an anti-parallel β-sheet rather than the α-helix and in disorder of the TonB box. Changes in conformation, but different in detail, at the N-terminus were observed in BtuB, FecA and FhuA complexes22,34,35, but no route for any conformational coupling with siderophore recognition was detected. We do see a correlation of movements between the binding site of the Fe3+-enterobactin molecule in the extracellular loops, the top of plug domain and the N-terminus. This suggests a route by which siderophore binding could be coupled to TonB engagement.

Binding of azotochelin and protochelin

Azotochelin and protochelin are two siderophores produced by Azotobacter vinelandii, an environmental Gram-negative bacterium. These two secondary metabolites are, respectively, a bis-catechol siderophore and a tris-catechol siderophore36 (Fig. 1a). It has been previously shown that in P. aeruginosa, PfeA is involved in the uptake of the ferric form of these siderophores, neither of which are produced by P. aeruginosa itself, thus P. aeruginosa can operate a siderophore ‘piracy strategy' to acquire iron28. Fe3+-enterobactin, Fe3+-azotochelin and Fe3+-protochelin coelute with PfeA on a size-exclusion column suggesting each of them are bound by PfeA (Supplementary Fig. 6). Crystal structures show that these two molecules bind in the same location as enterobactin (with less than 0.5 Å shift between the relative position of the iron atoms) and two of the catecholate rings occupy the same positions as rings II and III of enterobactin (Fig. 4; Supplementary Fig. 3c, d, Supplementary Fig. 4c, d). Hydrogen bonds between the catecholates and Ser479, Gly325 and Arg480 are conserved. In the azotochelin complex, a molecule of ethylene glycol from the crystallisation solution completes the coordination shell of the iron, and this molecule interacts with Gln482. The stacking interaction between Arg480 and catecholate II has been conserved. However, the side chain of Gln482 is unable to form a hydrogen bond with the aliphatic backbone of azotochelin, and is slightly displaced. The carboxylate group of the backbone is pointing towards the surface of the protein in the vicinity of Arg480 and Lys218. In protochelin, the presence of one supplementary carbon atom in the backbone between ring I and III perturbs the interaction with Arg480.

Fig. 4 PfeA recognises the catecholate siderophores azotochelin and protochelin. F O –F C electron density omit maps at 3 σ around Fe3+-azotochelin (a) and Fe3+-protochelin (b). Azotochelin is shown as sticks with carbon atoms coloured salmon, and protochelin carbon atoms are in green. Fe3+ are represented as orange spheres. Binding sites of Fe3+-azotochelin (c) and Fe3+-protochelin (d) are the same as Fe3+-enterobactin. Residues within 4.0 Å of the siderophores are displayed. Residues of the proteins are represented as sticks with carbon atoms coloured in yellow, nitrogen in dark blue and oxygen in red. Hydrogen bonds are shown as black broken lines. In c, the molecule of ethylene glycol completing the coordination shell of the iron is represented with white sticks Full size image

Site-directed mutagenesis and biochemical characterisation

We constructed four mutants R480A, Q482A, G324V and R480A-Q482A (double mutant) to validate the observed recognition site. Each of the mutant proteins was purified as described for the parent protein, and crystal structures of each were obtained. These structures were essentially identical to that of the parent protein, which confirmed that the mutations did not affect the folding of the protein (Supplementary Fig. 7a). Co-complexes with Fe3+-enterobactin were obtained by soaking for the R480A and Q482A mutants (Supplementary Fig. 3e, f, Supplementary Fig. 7b, c). Apart from the mutation itself and an additional water molecule in R480A, the mutant complexes were essentially identical except that the B-factors of the Fe3+-enterobactin molecules were higher reflecting a bigger mobility or a partial occupancy of the ligand. Neither G324V nor the double mutant were able to form a complex with Fe3+-enterobactin (Supplementary Fig. 3g, h). Unlike the parent protein, the R480A-Q482A double mutant does not coelute with Fe3+-enterobactin (Fig. 5a).

Fig. 5 The double-mutant R480A-Q482A abolishes Fe3+-enterobactin binding. a Unlike the wild-type protein (blue line), the double-mutant R480A-Q482A (red line) does not coelute with Fe3+-enterobactin. Proteins were incubated with the Fe3+-siderophore before being loaded on a S200 size-exclusion column. UV-visible spectra of PfeA shows a peak of absorption around 550 nm characteristic of the iron-siderophore complex. b Isothermal calorimetry titration of Fe3+-enterobactin with PfeA (blue) and mutants R480A-Q482A (red), G324V (cyan), R480A (green) and Q482A (black) Full size image

Titration of Fe3+-enterobactin into PfeA by isothermal titration calorimetry (ITC) reveals an unusual biphasic behaviour (Fig. 5b; Supplementary Fig. 8, Supplementary Data 1–10) that cannot be fitted by a simple one binding site model. This result can be fitted with a cooperative two binding sites per protein model. The sites have different affinities for the ligand, one with high affinity and highly favourable enthalpy and the second with lower affinity and unfavourable enthalpy. Although the biphasic nature of the isotherm was reproducible, the numerical values varied. For K1, the binding constant was 20 to 100 nM; for K2 190 to 120 μM. The same titration was carried out with G324V and R480A-Q482A, in both these mutants no binding was detected. Mutant Q482A showed biphasic isotherms, but with much reduced heat output and smaller K1 values. Mutant R480A showed a typical low affinity-binding curve profile. The weaker binding at the first site is consistent with the higher B-factor of the Fe3+-enterobactin molecule observed in the complex crystal structures.

Effects of PfeA mutation on iron uptake

To assess the effect of mutation in vivo on the transport and accumulation of iron, we used a P. aeruginosa strain in which the PfeA gene was deleted (∆pfeA). This knockout strain was transformed with a plasmid containing a gene coding for the double-mutant PfeAR480A-Q482A (∆pfeA(pMMBpfeAR480AQ482A)), and assessed with a 55Fe uptake assay. We also measured accumulation of iron in the wild-type PAO1 strain, ∆pfeA cells transformed with native PfeA protein (∆pfeA(pMMBpfeA)) and ∆pfeA cells with no plasmid. Cells were incubated with or without the presence of carbonyl cyanide m-chlorophenyl hydrazine (CCCP, an uncoupler of the proton-motive force known to inhibit all TonB-dependent transporters)37. In a first step, the mRNA levels of the PfeA protein and double mutant were determined to check the transcription levels of pfeA in all strains used for the 55Fe uptake assay.

The mRNA levels for pfeA gene were 160-fold and 100-fold higher for the two strains carrying the plasmids pMMBpfeA and pMMBpfeAR480AQ482A, respectively, compared with the wild-type strain PAO1 (Fig. 6a; Supplementary Data 11). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of outer-membrane preparations shows that both the native and double-mutant proteins are clearly present in the outer membrane of plasmid transformed cells (Supplementary Fig. 9). These data echo the heterologous expression of PfeA and mutants in E. coli used for the structural studies where the proteins are purified from the E. coli outer membrane at similar levels. Visual comparison of the SDS-PAGE gels (Supplementary Fig. 9) show much higher levels of both native and double-mutant proteins in the outer membrane of knockout cells (with corresponding plasmid) than the PfeA level in wild-type PAO1; this is consistent with the RT-qPCR data. We are therefore confident that the plasmid containing knockout cells, possess more than sufficient quantities of native and double-mutant PfeA proteins in their outer membrane to reliably report protein binding and transport activity.

Fig. 6 Effects of PfeA mutation in vivo. a Analysis of changes in the transcription of pfeA gene. PAO1 strain, its corresponding deletion mutant ∆pfeA and deletion mutants carrying the expression plasmid of PfeA wild-type (∆pfeA(pMMBpfeA) or the R480A-Q482A mutant (∆pfeA(pMMBpfeAR480AQ482A)) were grown in CAA medium supplemented with 10 µM enterobactin. The data were normalised relative to the reference gene uvrD, and are representative of three independent experiments performed in triplicate (n = 3). The results are given as a ratio between the values obtained for ∆pfeA, ∆pfeA(pMMBpfeA) and ∆pfeA(pMMBpfeAR480AQ482A) over those obtained for the PAO1 strain. b 55Fe-enterobactin binding at the bacterial surface. Cells were grown as in panel a, and were incubated for 15 min with 200 µM CCCP before initiation of transport assays by the addition of 500 nM 55Fe-enterobactin. After 30 min incubation, the radioactivity accumulated in the bacteria was counted. The results are expressed as pmol of 55Fe-enterobactin bound per ml of cells at an OD 600 of 1. The experiments have been repeated three times. c 55Fe-enterobactin binding and uptake. Cells were grown as in panel a, and were incubated with or without 200 µM CCCP before initiation of transport assays by the addition of 500 nM 55Fe-enterobactin. After 30 min incubations, the radioactivity accumulated in the bacteria was counted. The results are expressed as pmol of 55Fe-enterobactin bound and transported per ml of cells at an OD 600 of 1. The experiments have been repeated three times Full size image

CCCP treated cells (Fig. 6b), which report only 55Fe-siderophore binding since transport is blocked, show that the knockout cells transformed with pMMBpfeA plasmid exhibit much higher levels of binding when compared with wild-type PAO1. This would be expected from the much higher level of PfeA protein in the outer membrane of the knockout cells (with native PfeA) than in wild-type PAO1 observed by SDS-PAGE. Knockout cells lacking a plasmid for pfeA show the same low level of binding as wild-type PAO1. This background level reflects the presence of other catecholate-binding proteins (e.g., PirA, CirA) present in both knockout cells and wild-type PAO1. Knockout cells transformed with double-mutant PfeA (∆pfeA(pMMBpfeAR480AQ482A)) show the same (within error) background level of 55Fe binding. Thus, presence of double-mutant PfeA, even though expressed at a very high levels (greatly facilitating detection), does not show any detectable increase over knockout cells with no PfeA in the amount of enterobactin binding. Consequently, we conclude that the double mutant does not bind enterobactin in vivo, echoing the conclusion from the in vitro work.

Knockout cells that are not treated with CCCP report on binding and transport combined (Fig. 6c; Supplementary Fig. 10). In the knockout strain containing no plasmid, there is a small but measurable increase in the amount iron bound (compared to CCCP treated). This reflects the active transport by the other catecholate-binding proteins (e.g., PirA, CirA) and establishes the background level for the knockout system. Knockout cells expressing PfeA show a much larger increase in the amount of iron bound and a higher level of total iron than knockout cells with no plasmid. This indicates that native PfeA increases the iron level in the knockout strain because enterobactin is being actively taken up by the native plasmid encoded PfeA protein. Knockout cells transformed with the double-mutant PfeA gave essentially identical results to the knockout strain without any plasmid, that is the presence of significant levels of the double-mutant PfeA in outer membrane does not increase uptake over background. We conclude that the double mutant PfeA is not competent for the uptake of enterobactin in vivo. These data provide strong evidence that the site identified by crystallography is essential for binding and transport of Fe3+-enterobactin in vivo.

Computational approaches

A blind docking experiment using PfeA-Fe3+-enterobactin structure but with the Fe3+-enterobactin removed yielded three possible docking poses (arising from the three fold rotational symmetry) that matched the experimental complex within 0.5 Å of r.m.s.d., with an average predicted free energy of binding of −16.7 kcal.mol−1 (Supplementary Fig. 11). The R480A-Q482A double mutant gave the same poses, but with a 2 kcal.mol−1 smaller predicted free energy of binding. Analysis shows that for the double mutant, the lifetime of the Fe3+-enterobactin complex is reduced by four orders of magnitude compared with native (10–3.8 and 100.8 s, respectively) indicating only a transitory interaction. Docking with the native (apo) protein structure, that is without the changes seen upon Fe3+-enterobactin binding, yields a pose for Fe3+-enterobactin with a much lower binding free energy (than docking with loops changed) and with a different orientation (compared to the crystal structure) (Supplementary Fig. 11).

Molecular dynamics trajectories of both the apo and ferric-siderophore complex structures were subjected to an extensive statistical analysis of internal voids38. In both setups, we observed two dynamic voids located between the Fe3+-enterobactin binding site and the NL1/NL3 plug region (Fig. 7; Supplementary Fig. 12). The voids share the same residues with the internal cavity and gateway observed in the crystal structures. Although, as a result of the molecular dynamics, there are changes in positions of some of the residues that form these voids when compared with the crystal structures. Starting from the ferric-siderophore complex, the structure of the voids correlates with the hydrogen-bonding arrangement of Glu327. When Glu327 is bound to Arg100 of NL3, an arrangement resembling the gateway and internal cavity seen in the complex crystal structure is observed. An alternative arrangement, not seen in the crystal structure but in the calculation, shows Glu327 bound to Arg68 of NL1 (Fig. 7a–c). As a result, a merged ‘super' cavity is created that encompasses the gateway and the internal cavity. This super cavity has a volume compatible with the binding of Fe3+-enterobactin (> 500 Å3), and is closer to the plug region.

Fig. 7 Voids identified by molecular dynamics simulations. a Molecular graphics illustrating gateway (magenta) and internal cavity (green) controlled by exchange of Glu327 salt bridging partners in the complex simulations. The NL1/NL3 loops of the plug region are in yellow and the rest of the protein is in white. b Time series of the size of the voids identified below the Fe3+-enterobactin-binding site of PfeA. We note that they are covering almost the entire trajectory. In some parts of the trajectory, a significant expansion of the internal cavity is observed. c Plug-Glu327 salt bridges that control the dynamical behaviour of the internal cavity. We observe the correlation between the volume expansion and breaking and forming of the Arg68-Glu327 salt bridge Full size image

In the ferric-siderophore complex structure, we observed strong correlations between the structural changes that occur in the extracellular loops forming the binding site with the wider extracellular region and the intracellular region, notably structural changes in the periplasmic N-terminal TonB box region. Correlations are also found in the apo-structure but are less extensive (Fig. 8a, b), suggesting the presence of Fe3+-enterobactin is important. Since the correlations are modulated by hydrogen bonds, the hydrogen bonding correlations between NL1/NL3 residues in the plug and the rest of the protein were examined in detail (Fig. 8c, d). The hydrogen bonding correlation matrix reveals a clear segregation of the hydrogen bonds in the complex, but not in the apo simulations. Thus, the binding of Fe3+-enterobactin to PfeA creates two highly correlated groups of hydrogen bonds connecting extracellular loops to the plug domain (Fig. 8d) with Glu327 as one of the main participants.