The Streptococcus pyogenes Shr protein captures human hemoglobin using two structurally unique binding domains

Abstract

In order to proliferate and mount an infection, many bacterial pathogens need to acquire iron from their host. The most abundant iron source in the body is the oxygen transporter hemoglobin (Hb). Streptococcus pyogenes, a potentially lethal human pathogen, uses the Shr protein to capture Hb on the cell surface. Shr is an important virulence factor, yet the mechanism by which it captures Hb and acquires its heme is not well-understood. Here, we show using NMR and biochemical methods that Shr binds Hb using two related modules that were previously defined as domains of unknown function (DUF1533). These hemoglobin-interacting domains (HIDs), called HID1 and HID2, are autonomously folded and independently bind Hb. The 1.5 Å resolution crystal structure of HID2 revealed that it is a structurally unique Hb-binding domain. Mutagenesis studies revealed a conserved tyrosine in both HIDs that is essential for Hb binding. Our biochemical studies indicate that HID2 binds Hb with higher affinity than HID1 and that the Hb tetramer is engaged by two Shr receptors. NMR studies reveal the presence of a third autonomously folded domain between HID2 and a heme-binding NEAT1 domain, suggesting that this linker domain may position NEAT1 near Hb for heme capture.

Keywords: DUF1533; Shr; Streptococcus pyogenes (S. pyogenes); X-ray crystallography; bacterial pathogen; hemoglobin; isothermal titration calorimetry (ITC); nuclear magnetic resonance (NMR); receptor.

Figure 1.

Domain arrangement within Shr and the protein constructs used in this study. The Top panel shows the domains that have been identified in Shr. Previous studies have shown that Shr contains an Hb-binding NTR and a hemin-binding CTR. Polypeptides used in this study contain regions within the NTR and include: Shr^HID1 and Shr^HID2 that contain the first and second DUF1533 domains (renamed HIDs), respectively; Shr^L, which is predicted to contain several α-helices; and Shr^HID1–2, which contains both of the DUF1533 domains. The DUF1533 domains are HIDs based on findings reported in this paper.

Figure 2.

Hb binding studies by NMR. Panels showing the ¹H–¹⁵N HSQC spectra of Shr proteins in the presence and absence of Hb. a and b show the ¹H–¹⁵N HSQC spectra [¹⁵N]Shr^HID1 in the presence and absence of Hb (1:1 molar equivalent in tetramer units), respectively. c and d show the ¹H–¹⁵N HSQC spectra [¹⁵N]Shr^HID2 in the presence and absence of Hb (1:1 molar equivalent in tetramer units), respectively. e and f show the ¹H–¹⁵N HSQC spectra [¹⁵N]Shr^L in the presence and absence of Hb (1:1 molar equivalent in tetramer units), respectively. Only polypeptides containing the DUF1533 domains interact with Hb.

Figure 3.

Biochemical studies of Hb binding. a–d show representative ITC Hb binding data for Shr^HID1–2 (a), Shr^HID2 (b), Shr^ΔHID2 (c), and Shr^HID1 (d). In each titration experiment, Shr polypeptides were injected into a cell containing Hb. In each panel, at the top is shown the time course of the titration (black) and baseline (red). The bottom part of the panel shows the integrated isotherms (squares) and the curve fit (line). As is standard, the first data point in each experiment was eliminated prior to analysis. The fifth data point in the Shr^HID1 titration was also eliminated due to a spurious double peak. Origin software was used to analyze the data. e, SEC-MALS data defining receptor–Hb interactions. Elution profiles of Hb alone, Shr^HID1–2 alone, and Hb in combination with Shr^HID1–2. Refractometer voltage is indicated on the left y axis, and the corresponding trace is shown in black. The elution volume is shown on the x axis. ASTRA software was used to analyze the data. The molecular weight is indicated on the right y axis. Interestingly, the measured Hb molecular weight is slightly smaller than its predicted molecular mass of 64.5 kDa based on its primary sequence, a discrepancy that has also been observed by others (65, 66). f and g, sedimentation equilibrium data defining receptor–Hb interactions. The panel shows profiles of Shr^HID2 alone (gray) at 26,000 rpm, as well as Hb alone (red) and in complex with Shr^HID2 (blue) at a 1:45 ratio of Hb0.1 (heme basis)/Shr^HID2 at 13,000 rpm. The top panels show the residuals of the fit of the experimental data, and the bottom panels show the absorbance readings on the y axis versus the radial position on the x axis. Circles and curved black lines correspond to experimental data and calculated fits to binding models described in the text, respectively.

Figure 4.

Hb binding competition experiment. NMR was used to demonstrate that the isolated HIDs domain can compete for the same site on Hb. Data are similar to that shown in Fig. 2. The panels show the ¹H–¹⁵N HSQC spectra of [¹⁵N]Shr^HID1. a shows the spectrum of the [¹⁵N]Shr^HID1 protein in isolation, and b shows its spectrum in the presence of 2-fold excess of Hb (heme basis). c and d are similar to b. They show the spectrum of a 2:1 (c) and a 4:1 (d) equivalent of unlabeled Shr^HID2/[¹⁵N]Shr^HID1 to the sample.

Figure 5.

Assignments and dynamics data on Shr^HID2. a, backbone assignments of Shr^HID2 overlaid onto the ¹H–¹⁵N HSQC spectrum. Left, entire HSQC spectrum is shown. Right, inset shows an enlarged view of the center, which has the greatest amount of spectral overlap. Of the Shr^HID2 construct, ∼94% of the residues were assigned. Unassigned residues correspond to two prolines, two residues at the N terminus, and five residues scattered throughout the domain. b, {¹H} ¹⁵N heteronuclear NOE values (on the y axis) shown on a per residue basis (on the x axis). Residues with missing heteronuclear NOE values were eliminated from the analysis due to either missing backbone assignments or severe spectral overlap. Shown are the average and standard deviation of three replicates.

Figure 6.

Crystal structure of Shr^ΔHID2. a, cartoon representation of the two Shr^ΔHID2 molecules in the asymmetric unit. b, chain B of the crystal structure with secondary structure elements labeled, and shown in red is the conserved tyrosine critical for Hb binding. c, electrostatic potential calculated using PyMOL, and shown is the same face of the domain as in b. Areas shaded in red and blue correspond to negative and positive potentials, respectively.

Figure 7.

Domain topology of Shr^ΔHID2versus NEAT domains. a and b, secondary structural elements of Shr^ΔHID2 showing the domain topology (a) compared with that of the canonical Hb-binding NEAT domains from S. aureus (b). Both domains are colored purple to red from the N to C terminus, respectively.

Figure 8.

Hb titration experiments with Shr^HID1 and Shr^HID2 mutants by NMR. a and b, normalized height of peaks in the ¹H–¹⁵N HSQC spectra (y axis) of [¹⁵N]Shr^HID2 and [¹⁵N]Shr^HID1 WT (black) as well as [¹⁵N]Shr^HID2-Y197A and [¹⁵N]Shr^HID1-Y55A (green) upon titrating the sample with Hb (x axis). Individual data points represent the average height of peaks used in the analysis, and error bars represent the standard deviation of those peaks used to calculate the average. Data points were normalized to the corresponding ¹H–¹⁵N HSQC spectrum prior to adding Hb.