Functionally distinct NEAT (NEAr Transporter) domains within the Staphylococcus aureus IsdH/HarA protein extract heme from methemoglobin

Abstract

The pathogen Staphylococcus aureus uses iron-regulated surface determinant (Isd) proteins to scavenge the essential nutrient iron from host hemoproteins. The IsdH protein (also known as HarA) is a receptor for hemoglobin (Hb), haptoglobin (Hp), and the Hb-Hp complex. It contains three NEAT (NEAr Transporter) domains: IsdH(N1), IsdH(N2), and IsdH(N3). Here we show that they have different functions; IsdH(N1) binds Hb and Hp, whereas IsdH(N3) captures heme that is released from Hb. The staphylococcal IsdB protein also functions as an Hb receptor. Primary sequence homology to IsdH indicates that it will also employ functionally distinct NEAT domains to bind heme and Hb. We have used site-directed mutagenesis and surface plasmon resonance methods to localize the Hp and Hb binding surface on IsdH(N1). High affinity binding to these structurally unrelated proteins requires residues located within a conserved aromatic motif that is positioned at the end of the beta-barrel structure. Interestingly, this site is quite malleable, as other NEAT domains use it to bind heme. We also demonstrate that the IsdC NEAT domain can capture heme directly from Hb, suggesting that there are multiple pathways for heme transfer across the cell wall.

FIGURE 1.

Comparison of the NEAT domains in S. aureus. A, schematic of the IsdB and IsdH proteins showing that they contain two and three NEAT domains, respectively. Domains are shaded based on their relatedness. Domains shaded gray share 46-65% primary sequence identity (IsdH^N1, IsdH^N2, and IsdB^N1). Non-shaded domains share 49% primary sequence identity (IsdH^N3 and IsdB^N2). Based on studies of the isolated NEAT domains within IsdH, the gray- and non-shaded domains bind Hb and heme, respectively. Both proteins also contain a cell wall sorting signal motif at their C termini that is covalently attached to the cell wall by the SrtA sortase enzyme. B, sequence alignment of related Hb binding NEAT domains within IsdH and IsdB: IsdH^N1, IsdH^N2, and IsdB^N1. These domains are closely related to one another and share 46-65% sequence identity. Residues mutated in this study are indicated with an asterisk, and amino acids are enclosed in a box if their mutation to alanine completely disrupts Hp binding and/or reduces the affinity of IsdH^N1 for Hb by at least ×50-fold. The aromatic motif diagnostic of NEAT domains that bind Hb is underlined. The secondary structure of IsdH^N1 is shown above the primary sequence. Completely conserved residues are in bold. Only the IsdH^N1 and IsdH^N2 domains have thus far been shown to bind Hb, whereas the IsdB^N1 domain alone has yet to be tested. C, sequence alignment of the IsdH^N3 and IsdB^N2 NEAT domains, which share 49% sequence identity. The sequences of the distantly related IsdA and IsdC domains are also shown because they bind to heme. Positions within the primary sequence that are within 3.5 Å of the heme in the NMR and crystal structures of the IsdC-heme and IsdA-heme complexes are underlined. The invariant tyrosine residues that coordinate the iron atom of the heme in these structures is enclosed in a box. Residues that are completely conserved in IsdH^N3 and IsdB^N2 are in bold.

FIGURE 2.

SPR measurements of IsdH^N1 binding to MetHb and Hp. A, representative SPR data of wild-type IsdH^N1 binding to immobilized MetHb. The panel shows an overlay of several experiments in which the concentration of IsdH^N1 was varied (0, 100, 200, 300, 400, 500, 1000, 2000, and 4000 nm). Each curve is a plot of the RU as a function of time. B, representative binding curve of the wild-type IsdH^N1 for immobilized MetHb. The curve was generated from the data in panel A by plotting the RU value measured 1 s before dissociation for each concentration of IsdH^N1. The solid line shows the best fit of this data to obtain the K_D of binding. C, representative SPR data of wild-type IsdH^N1 binding to immobilized Hp. The results of six independent experiments are superimposed and differ in the concentration of concentration of IsdH^N1 (0, 100,200,300,400,500, and 1000 nm IsdH^N1). D, representative binding curve of wild-type IsdH^N1 for immobilized Hp by plotting the RU value measured 1 s before dissociation for each concentration of IsdH^N1.

FIGURE 3.

The IsdH^N3 NEAT domain binds heme. A, UV spectrum of purified IsdH^N3 that show characteristics of a heme-binding protein. These include a Soret band at 402 nm along with additional Q-band peaks at 511, 548, and 625 nm. Abs, absorbance. B, quantitative measurement of protoporphyrin binding using a fluorescence assay. The panel shows a plot of ZnPPIX fluorescence emission at 585 nm as a function of IsdH^N3 added. The solid line shows a best fit of this data which yields a K_D of ZnPPIX binding to IsdH^N3 of 2.8 ± 0.1 μm. C, an ELISA assay showing that the IsdH^N1 NEAT domain binds MetHb, whereas the IsdH^N3 does not. IsdH^N3 binding to a range of other proteins was also tested, but no interaction could be detected.

FIGURE 4.

Transfer of heme from MetHb to IsdH^N3 and IsdC. A, bar graph showing the percentage of heme recovered after MetHb is incubation with talon beads for 120 min. The negative control indicates that the talon beads were naked. IsdHN3 and IsdC indicate that the talon beads used in this experiment were preincubated with the His-tagged versions of these proteins. B, quantitative measurement of the rate of heme transfer from MetHb to IsdH^N3. The open circles represent heme transfer from MetHb to IsdH^N3 as a function of time. The closed circles indicate transfer from MetHb when it is bound to IsdH^N1 in the IsdH^N1-MetHb complex. The solid line is the best fit of these data and was used to extract the rate of transfer (described under Heme Transfer Assay (see “Experimental Procedures”)). C, quantitative measurement of heme transfer from MetHb to IsdC. Results are as described in panel B, except that His-IsdC protein was used as the receptor for heme. The open and closed squares represent heme transfer from MetHb and the IsdH^N1-MetHb complex, respectively.

FIGURE 5.

A common surface on the IsdH^N1 NEAT domain binds haptoglobin and methemoglobin. A, ribbon drawing of the solution structure of IsdH^N1. Strands of β sheet are gray and are indicated by arrows. The three loops involved in protein binding are indicated. B, solvent-exposed surface of IsdH^N1 color coded to show the effects of amino acid mutations on MetHb binding. Color coding key: red, > 50× reduction or no detectable binding; dark purple, 10-11× reduction; pink, 2-4× reduction; tan, no significant effect on binding (<2× reduction). C, solvent-exposed surface of IsdH^N1 color-coded to show the effects of amino acid mutations on MetHb binding. Color coding is as described in panel B. The view of the structure is identical in all of the panels.

FIGURE 6.

Schematic showing how the IsdH protein may capture and transfer heme from hemoglobin. Step 1, MetHb is tethered to IsdH via the N-terminal IsdH^N1 and IsdH^N2 NEAT domains. MetHb is shown as two hexagons representing the α and β globin chains. Heme molecules are represented by five-pointed stars. Step 2, heme is released from MetHb. Our data indicates that this process is passive when NEAT domains are in isolation. Step 3, heme diffuses and is captured by the C-terminal IsdH^N3 NEAT domain. Step 4, heme is released from IsdH^N3 and subsequently captured by IsdC located within the cell wall. Our results also indicate that IsdC can directly capture heme that is passively released from MetHb (dashed line). Note that IsdH and IsdC are covalently attached to the cell wall by the SrtA and SrtB sortases, respectively.