Structure-function analyses reveal key features in Staphylococcus aureus IsdB-associated unfolding of the heme-binding pocket of human hemoglobin

Abstract

IsdB is a receptor on the surface of the bacterial pathogen Staphylococcus aureus that extracts heme from hemoglobin (Hb) to enable growth on Hb as a sole iron source. IsdB is critically important both for in vitro growth on Hb and in infection models and is also highly up-regulated in blood, serum, and tissue infection models, indicating a key role of this receptor in bacterial virulence. However, structural information for IsdB is limited. We present here a crystal structure of a complex between human Hb and IsdB. In this complex, the α subunits of Hb are refolded with the heme displaced to the interface with IsdB. We also observe that atypical residues of Hb, His⁵⁸ and His⁸⁹ of αHb, coordinate to the heme iron, which is poised for transfer into the heme-binding pocket of IsdB. Moreover, the porphyrin ring interacts with IsdB residues Tyr⁴⁴⁰ and Tyr⁴⁴⁴ Previously, Tyr⁴⁴⁰ was observed to coordinate heme iron in an IsdB·heme complex structure. A Y440F/Y444F IsdB variant we produced was defective in heme transfer yet formed a stable complex with Hb (K_d = 6 ± 2 μm) in solution with spectroscopic features of the bis-His species observed in the crystal structure. Haptoglobin binds to a distinct site on Hb to inhibit heme transfer to IsdB and growth of S. aureus, and a ternary complex of IsdB·Hb·Hp was observed. We propose a model for IsdB heme transfer from Hb that involves unfolding of Hb and heme iron ligand exchange.

Keywords: Staphylococcus aureus (S. aureus); crystal structure; heme; hemoglobin; iron.

Figure 1.

The IsdB^N1N2·Hb complex. Left panel, components of the IsdB^N1N2·Hb crystal separated by SDS-PAGE. Bands of the expected molecular weight of Hb and IsdB^N1N2 were detected along with a ∼19-kDa band presumed to be an IsdB fragment. Right panel, crystal structure of the overall IsdB^N1N2·Hb complex. IsdB molecules are colored in shades of blue, αHb molecules are colored in beige, and βHb molecules are colored in dark orange. Heme moieties in the αHb chains are shown as green sticks.

Figure 2.

Alterations in Hb polypeptide chain structure in the complex with IsdB. A, left panel, a single copy of αHb (beige) interacting with a single copy of IsdB^N1N2 (dark blue). IsdB^N2 directly interacts with the Hb heme and heme pocket. The linker is a three-helix bundle between the two NEAT domains. IsdB^N1 binds onto the opposite face of αHb. Right panel, the conformation of the αHb C–D loop is significantly altered upon IsdB binding. B, stereo view of the interaction with IsdB, which results in the αHb F helix becoming highly unwound. The αHb chain of oxyHb (PDB code 2DN1; green) is overlaid to demonstrate the original state of the helix. The heme is illustrated in blue sticks with heme iron in red. C, stereo view of the interaction between βHb (orange) and IsdB^N1 (cyan). The βHb chain of oxyHb with associated heme (PDB code 2DN1; gray) is overlaid. The orientation of the molecules is similar to that of A. Density for the βHb F helix and heme is absent, as are the IsdB linker and NEAT2 domains.

Figure 3.

Binding of IsdB^N1N2 to Hb induces major changes in heme environment. A, stereo view of an F_o − F_c omit positive difference map (green) of the heme in the IsdB^N1N2·αHb interface contoured at 3σ (refined with torsion-based simulated annealing). An anomalous density map (pink) contoured at 3σ is overlaid to unambiguously support correct placement of the heme. B, the complex structure is overlaid with the structure of the αHb chain from the oxyHb structure (PDB code 2DN1). The 2DN1 αHb structure is shown in green; αHb and IsdB^N1N2 from the IsdB^N1N2·Hb structure are shown in beige and blue, respectively. Relevant amino acid side chains are shown as sticks. Only the heme moiety from the structure presented here is shown (dark green). C, the IsdB heme pocket is positioned to accept αHb-heme. IsdB and αHb residues situated at the binding interface near the heme are shown as blue and beige sticks, respectively. The remainder of the structure is shown as cartoons. The αHb-heme is shown in green, with the heme iron (coordinated by αHis⁵⁸ and αHis⁸⁹) shown as an orange sphere.

Figure 4.

Electronic spectra of IsdB^YFYF combined with heme and Hb. A, addition of excess IsdB^YFYF to metHb results in a distinctly altered spectrum (blue); the heme spectrum alone (red), metHb spectrum alone (purple), the mixture of IsdB^YFYF with heme (green), and reaction of wild-type IsdB^N1N2 with metHb (orange) are shown for comparison. AU, absorbance units. B, a closer look at the visible region of the spectra presented in A.

Figure 5.

Titration of metHb with IsdB^YFYF resulted in dose-dependent, saturable changes in electronic spectra. 4 μn metHb was titrated with increments of 1 μm of apo-IsdB^YFYF from 1 to 9 μm at 22 °C; spectral changes were monitored in a conventional spectrophotometer. Spectra shown are the average of three independent replicates. A, overall spectral changes accompanying the titration of IsdB^YFYF into metHb. The spectrum of metHb alone is shown in blue, the final titration spectrum is shown in red, and each gray line represents an intermediate titration spectrum in 1 μm increments. AU, absorbance units. B, expansion of spectra in the α/β region of the spectra shown in A. C, the change in absorption at 410 nm plotted against the concentration of IsdB^YFYF for each titration point. Each point represents the mean and standard error of three replicates. The dotted line represents a linear fit to the first five titration points to indicate the concentration of IsdB^YFYF where a plateau begins.

Figure 6.

Heme transfer kinetics from metHb to IsdB^N1N2. A and B, electronic spectra collected with a stopped-flow spectrophotometer equipped with a photodiode array. A, spectra recorded over 15 s after mixing of 2 μn metHb with 20 μm IsdB^N1N2. B, an increase in the Soret peak was observed over the first ∼60 ms, from ∼350–420 nm. AU, absorbance units. C, a representative single-wavelength (428 nm) stopped-flow spectroscopy experiment where 1 μn metHb was mixed with 17 μm IsdB^N1N2. The gray bars represent the standard error of four replicates, and the black curve is a fit to a single exponential equation. The residuals for the experiment are in the inset. D, the observed transfer rate (k_obs) from 1 μn metHb is plotted as a function of IsdB^N1N2 concentration. The line is a hyperbolic fit assuming a two-step reaction model. Each point represents the mean, and the bars are the standard errors of four replicates. The residuals of the data to the model are in the inset.

Figure 7.

Inhibition of heme transfer from metHb by Hp. A, preincubating 2 μn metHb (blue) with 2 mg/ml mixed-serotype Hp resulted in metHb-like spectra (not shown). The addition of 10 μm IsdB^N1N2 (green) resulted in a modest increase in the Soret peak. The reaction of 2 μn metHb with 10 μm IsdB^N1N2 (in the absence of Hp) is shown for comparison (red). AU, absorbance units. B, 2 μn metHb was preincubated with decreasing amounts of Hp, as indicated, followed by the addition of 10 μm IsdB^N1N2. Spectra for each reaction were recorded within 20 s of mixing and did not change within 5 min. C, SDS-PAGE separation of nickel-nitrilotriacetic acid bead pulldown of His₆-IsdB^N1N2, Hp, and metHb. 20 μm His₆-IsdB^N1N2 was used as bait to pull down 20 μn metHb and/or ∼20 μm Hp. His₆-IsdB^N1N2 could bind nickel beads alone (lane 1), whereas metHb and Hp could not (lanes 2 and 3, respectively). His₆-IsdB^N1N2 pulls down metHb (lane 4), but not Hp (lane 5). When metHb is added to nickel beads mixed with His₆-IsdB^N1N2 and Hp, all three species are pulled down (lane 6). 1 μg of Hp is shown in lane 7, for reference. Although Hp runs at nearly the same position on the gel as His₆-IsdB^N1N2, two separate bands in lane 6 are distinguished, largely because of their differential staining (Hp is glycosylated, affecting staining by Coomassie dye).

Figure 8.

Growth of S. aureus for 16 h on iron-depleted RPMI medium supplemented with 200 nn Hb or 2 μn heme as the sole iron sources. Increasing concentrations of Hp inhibited growth of S. aureus on medium supplemented with Hb but not on heme. Each bar is the average of three independent growth experiments conducted on a Bioscreen C, each with three technical replicates.

Figure 9.

A model of the heme extraction pathway. A, heme positions observed in αHb, the IsdB^N1N2·Hb complex, and isolated IsdB. The αHb chain of oxyHb (PDB code 2DN1; beige) overlaid on top of the complex αHb (dark blue) is used to represent the pretransfer heme position in uncomplexed, folded Hb. The heme-bound form of IsdB^N2 (PDB code 3RTL heme conformation A; pink) overlaid on top of the complex IsdB^N2 domain (cyan) represents the completed heme transfer reaction. B, top-down view of the positional changes of the heme molecule shown in A. The heme iron in the complex structure (dark blue) is ∼5 Å away from both the initial and final heme iron positions.

Figure 10.

A stereo overlay of the Hp·Hb structure with the IsdB^N1N2·Hb structure. Shown is superposition of porcine Hp·Hb (PDB code 4F4O) with one of the α/β Hb dimers of the IsdB^N1N2·Hb structure. Hp binds at a distinct site from IsdB. Hp is shown in purple, and IsdB^N1N2 and IsdB^N1 are shown in dark blue and light blue, respectively. The αΗβ and βHb subunits are shown in beige and orange, respectively, with the αHb heme shown as bright green sticks.