The siderocalin/enterobactin interaction: a link between mammalian immunity and bacterial iron transport

Abstract

The siderophore enterobactin (Ent) is produced by enteric bacteria to mediate iron uptake. Ent scavenges iron and is taken up by the bacteria as the highly stable ferric complex [Fe (III)(Ent)] (3-). This complex is also a specific target of the mammalian innate immune system protein, Siderocalin (Scn), which acts as an antibacterial agent by specifically sequestering siderophores and their ferric complexes during infection. Recent literature suggesting that Scn may also be involved in cellular iron transport has increased the importance of understanding the mechanism of siderophore interception and clearance by Scn; Scn is observed to release iron in acidic endosomes and [Fe (III)(Ent)] (3-) is known to undergo a change from catecholate to salicylate coordination in acidic conditions, which is predicted to be sterically incompatible with the Scn binding pocket (also referred to as the calyx). To investigate the interactions between the ferric Ent complex and Scn at different pH values, two recombinant forms of Scn with mutations in three residues lining the calyx were prepared: Scn-W79A/R81A and Scn-Y106F. Binding studies and crystal structures of the Scn-W79A/R81A:[Fe (III)(Ent)] (3-) and Scn-Y106F:[Fe (III)(Ent)] (3-) complexes confirm that such mutations do not affect the overall conformation of the protein but do weaken significantly its affinity for [Fe (III)(Ent)] (3-). Fluorescence, UV-vis, and EXAFS spectroscopies were used to determine Scn/siderophore dissociation constants and to characterize the coordination mode of iron over a wide pH range, in the presence of both mutant proteins and synthetic salicylate analogues of Ent. While Scn binding hinders salicylate coordination transformation, strong acidification results in the release of iron and degraded siderophore. Iron release may therefore result from a combination of Ent degradation and coordination change.

Figure 1

Complexation of ferric ion by the siderophore enterobactin (top, the iron coordinating atoms are indicated in red) and specific binding of the ferric complex of enterobactin [Fe^III(Ent)]³⁻ by the mammalian protein Scn (bottom).

Figure 2

Conformation changes undergone by ferric- (top) and apo- (bottom) enterobactin upon protonation. The coordination of the iron center shifts from catecholate (top left) to salicylate (top right) as the complex is protonated, and the catecholate rings rotate around the amide bonds as the free ligand is protonated.

Figure 3

Fluorescence quenching analyses of the binding of the wild type Scn (Scn-WT) and the two mutants Scn-Y106F and Scn-W79A/R81A with the ferric complexes of the salicylate analogs of Ent: [Fe^III(SERSAM)]⁰ (top) and [Fe^III(SER(3M)SAM)]⁰ (middle), at pH 5.5 (the iron-coordinating oxygen atoms are indicated in red). Bottom: fluorescence quenching analyses of Scn-WT binding with the ligands SERSAM and (SER(3M)SAM) at pH 7.4. Symbols give the fluorescence data at 340 nm, and lines give the calculated fits to a one-binding site model.

Figure 4

Fluorescence quenching analyses of the binding of the wild type Scn (Scn-WT) and the two mutants Scn-Y106F and Scn-W79A/R81A with ferric (top) and apo (bottom) enterobactin. Symbols give the fluorescence data at 340 nm, and lines give the calculated fits to a one-binding site model.

Figure 5

Removal of the steric clashes in Scn causes changes in the observed siderophore binding. Although [Fe^III(Ent)]³⁻ is broken down in the wild type structure (top left panel) the break down products arrange themselves similar to that of the modeled intact complex (top right panel). In the Scn-W79A/R81A structure, removal of the key residues W79 and R81 allows for the binding of 2,3-DHBS in an inverted orientation (bottom left panel). In the Scn-Y106F structure, only one 2,3-DHBS fragment is bound (bottom right panel). The surface representation of the protein (top left and bottom panels) shows the electrostatic potentials for the surface of the protein (blue positively charged, white neutral, and red negatively charged). Inserts: co-crystals of the corresponding protein:ferric-siderophore adducts (colored for Scn and Scn-W79A/R81A; colorless for Scn-Y106F).

Figure 6

Fluorescence quenching analyses of wild type Scn (Scn-WT) binding with ferric- (top) and apo- (bottom) enterobactin as a function of pH. Symbols give the fluorescence data at 340 nm, and lines give the calculated fits to a one-binding site model.

Figure 7

Fluorescence pH-titration of Scn:[Fe^III(Ent)]³⁻. The protein fluorescence (340 nm) is first quenched by addition of ligand and increases upon acidification.

Figure 8

Spectrophotometric titration of Scn:[Fe^III(Ent)]³⁻ by HCl in water. I = 0.1 (KCl), T = 25.0 °C, l = 1 cm. Spectra are corrected for dilution, approximately 50% of the data is shown for clarity; 1 (pH 6.68); 2 (pH 6.12); 3 (pH 5.27); 4 (pH 4.08); 5 (pH 3.58); 6 (pH 2.70); 7 (pH 2.23); 8 (pH 1.84).

Figure 9

Fourier transforms of Scn-bound [Fe^III(Ent)]³⁻ at pH values from 7.4 to 1.5.

Figure 10

HPLC traces of sample solution filtrates after pH adjustment and ultracentrifugation through 10,000 kDa cutoff filters: Scn at pH 7.4 (a), Scn:[Fe^III(Ent)]³⁻ at pH 7.4 (b), Scn:[Fe^III(Ent)]³⁻ at pH 2.5 (c), [Fe^III(Ent)]³⁻ at pH 7.4 (d), [Fe^III(Ent)]³⁻ at pH 2.5 (e). The following retention times correspond to: 1.4 min, injection peak; 4.9 min, 2,3-DHBS; 16.3 min, Ent.