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.

Introduction

Streptococcus pyogenes (Group A Streptococcus) each year causes an estimated 700 million infections worldwide (1). Although most of these infections lead to nonlife-threatening acute pharyngitis, many are lethal and cause ∼500,000 deaths annually (2). These more severe and invasive infections have a high mortality rate (∼30%) and include necrotizing fasciitis and streptococcal toxic shock syndrome (3, 4). Infections caused by this deadly microbe are presently among the top 10 causes of death from an infectious disease worldwide (5). S. pyogenes and other pathogenic bacteria require iron to proliferate, as it is a versatile metal that functions as a key biocatalyst and electron carrier in enzymes that mediate metabolism (6). During infections, iron is frequently foraged from human hemoglobin (Hb),³ which contains ∼75–80% of the body's total iron content in the form of heme (iron–protoporphyrin IX). Recent studies have shown that S. pyogenes uses an array of surface and membrane-associated proteins to acquire Hb's heme. Understanding how these proteins function at a molecular level is of fundamental importance and could facilitate the discovery of new anti-infective agents that work by limiting microbial access to iron.

Surface and membrane-associated proteins are used by S. pyogenes to capture heme. The streptococcal hemoprotein receptor (Shr) is displayed on the bacterium's surface, where it binds to Hb and acquires the oxidized form of heme (called hemin) (7–11). The Shr Hb receptor is an important virulence factor, as Δshr mutant strains of S. pyogenes exhibit reduced growth in human blood and attenuated virulence in murine and zebrafish models of infection (10, 12). In vitro, Shr transfers hemin directly to Shp, a cell wall–associated hemoprotein that uses a bis-methionine ligation mechanism to interact with hemin (13–15). Shp then passes hemin to HtsA/SiaA, the lipoprotein component of the ABC transporter HtsABC/SiaABC (16). A binary Shp–HtsA complex mediates this transfer step, in which the methionine axial ligands in Shp are displaced by methionine and histidine axial ligands within HtsA (17–21). This process occurs very rapidly, >100,000 times faster than the rate at which Shp spontaneously releases hemin into the solvent. Hemin is then presumably transported across the membrane into the bacterial cytoplasm where it is degraded by a yet to be identified oxygenase(s) that releases hemin's iron for use by the microbe (6). Although hemin transfer from Shp to HtsA/SiaA has been extensively studied by Lei and co-workers (20), the first step in the pathway, the extraction of hemin from human Hb by Shr, is less well-understood and the focus of this study.

Many Gram-positive bacterial pathogens display Hb receptors that contain near–iron transport (NEAT) domains (22, 23). NEAT domains have been shown to bind Hb, hemin, and in many instances are able to rapidly transfer hemin among one another (24–35). Hemin acquisition by Staphylococcus aureus has been studied extensively. This microbe uses an array of iron-regulated surface determinant (Isd) proteins that each contain one or more NEAT domains (24, 29, 36–40). Hb is captured on the cell surface by the closely related IsdB and IsdH proteins (41). Our studies have shown that IsdH captures and extracts hemin from Hb using a conserved tri-domain unit that contains two NEAT domains, called N2 and N3, that are separated by a helical linker domain (42). As compared with the rate of hemin released into the solvent by Hb, binding of the tri-domain unit accelerates hemin release 13,400-fold (43). Atomic structures of IsdH–Hb complexes reveal that its N2 and N3 NEAT domains adopt related β-sandwich structures that are decorated by short helices, whereas the intervening linker domain adopts a three-helix bundle (44, 45). The N2 domain engages Hb's A-helix with high affinity, enabling the helical and N3 domains to distort Hb's F-helix, thereby promoting hemin transfer to IsdH's N3 domain (43, 45, 46). IsdB shares significant primary sequence homology with IsdH and also contains a tri-domain unit that captures hemin from Hb through a similar mechanism (31, 34, 42, 47–50). Other Gram-positive pathogens use NEAT domains to bind hemin and/or Hb, and include among others Listeria monocytogenes, Bacillus anthracis, Bacillus cereus, and S. pyogenes (23). However, the mechanisms used by these domains to capture hemin from Hb may be distinct from that in IsdH and IsdB, because in some instances a single NEAT domain has been proposed to bind to both Hb and heme (51–54). Notable examples are IsdX1, IsdX2, and Hal in which single NEAT domains from these proteins have been reported to bind Hb and extract hemin from Hb (52–54). However, complexes of these NEAT domains bound to Hb have not been visualized at atomic resolution.

Unlike other Hb receptors in Gram-positive bacteria, the S. pyogenes Shr protein does not use NEAT domains to engage Hb. Instead, it binds Hb via an N-terminal region (NTR) that is predicted to contain two domains of unknown function, called DUF1533 domains (Fig. 1) (8, 11). Released hemin is then bound by residues located within Shr's C-terminal region (CTR) (residues 365–1275), which contains two hemin-binding NEAT domains (NEAT1 and NEAT2) that are separated from one another by a series of leucine-rich repeats (8). Interestingly, similar to the staphylococcal IsdB and IsdH proteins, transfer experiments have shown that the isolated Shr protein increases the rate of hemin release from Hb in vitro, suggesting that the domains within the receptor function synergistically to extract hemin (9). The NEAT domains within Shr can rapidly transfer hemin among one another, but only NEAT1 is able to efficiently transfer hemin to Shp (55). These findings led to the suggestion that NEAT2 stores hemin near the cell surface (55).

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.

Here, we show that Shr uses two related DUF1533 domains to engage Hb, which we rename Hb-interacting domains (HIDs). Biochemical studies and the atomic structure of a HID reveal that it is a structurally novel Hb-binding module that employs a conserved tyrosine residue for Hb recognition. Biochemical studies indicate that the HIDs within Shr bind Hb with differing affinities and suggest that each engages a single globin subunit. Interestingly, NMR studies reveal the presence of a third autonomously folded domain that is located between the HID2 and NEAT1 domains. The implications of this arrangement in hemin acquisition from Hb are discussed.

Results

Hb-interacting N-terminal region within Shr contains three autonomously folded domains

Previous studies have shown the S. pyogenes Shr protein interacts with Hb via residues located at its N terminus (8). This N-terminal region is formed by residues Lys-25–Val-364 and is hereafter referred to as the NTR (Fig. 1). An inspection of its primary sequence suggests that the NTR contains three folded regions. These include two domains of unknown function (DUF1533 domains) that are formed by residues Gly-61–Phe-123 and Ile-203–Val-269 (8), as well as residues Val-299–Ser-358 that are predicted to form several α-helices. Constructs containing residues Gly-61–Phe-123 and Ile-203–Val-269 were insoluble. Secondary structural prediction algorithms such as PSIPRED (56) and JPred (57) were used to guide the design of additional constructs until highly soluble ones were obtained and characterized by NMR: Shr^HID1, residues Ser-26–Lys-148 that correspond to the first DUF1533 domain; Shr^HID2, residues Lys-155–Gln-285 that correspond to the second DUF1533 domain; and Shr^L, residues Ser-294–Val-364 that contain the predicted α-helical region in Shr that links Shr^HID2 to the first NEAT domain (Fig. 1). In each case, constructs included at least two residues beyond the predicted secondary structure elements at both the N and C termini. If ambiguous, extra residues were included to ensure that no potentially important structural elements were truncated. Each polypeptide was uniformly ¹⁵N-labeled and purified, and its ¹H–¹⁵N HSQC spectrum was recorded (Fig. 2, a, c, and e). All three polypeptides adopt a folded structure, as the cross-peaks in each spectrum are well-dispersed and have appropriate line widths. Based on the Pfam prediction for their domain boundaries, the two DUF1533 domains within the NTR are separated by ∼80 amino acids. To determine whether the domains interact with one another, a longer polypeptide fragment containing both domains was characterized by NMR (Shr^HID1–2, consisting of residues Ser-26–Gln-285). Superposition of the NMR spectra of [¹⁵N]Shr^HID1 and [¹⁵N]Shr^HID2 onto the spectrum of [¹⁵N]Shr^HID1–2 reveals that the cross-peaks have similar chemical shifts (Fig. S1). Thus, in the context of the longer Shr^HID1–2 polypeptide, the Shr^HID1 and Shr^HID2 domains do not significantly interact with one another, and they exhibit the same structural fold as they do in isolation. The Shr^HID2 and Shr^L domains also do not interact with one another, as the NMR spectra of a polypeptide containing both of these domains ([¹⁵N]Shr^HID2-L, residues Gly-61–Asn-368) can be recapitulated by summing the spectra of the isolated [¹⁵N]Shr^HID2 and [¹⁵N]Shr^L domains (Fig. S1). We conclude from the NMR data that the Hb-interacting region within Shr contains three independently folded and noninteracting structural domains, two DUF1533 domains, and a linker domain that is predicted to contain several α-helices.

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.

DUF1533 domains bind Hb

We used NMR to determine whether the domains within the NTR interacted with Hb. The ¹H–¹⁵N HSQC spectrum of each ¹⁵N-labeled domain was recorded in the presence or absence of Hb (1:1 stoichiometry based on tetramer units) (Fig. 2). Upon adding Hb to a solution containing [¹⁵N]Shr^HID1, a massive loss of signal occurs indicative of complex formation. This is caused by increased T₂ relaxation that occurs because of the significantly larger molecular weight of the complex, and possibly as a result of intermediate conformational exchange on the chemical shift time scale. A similar result is obtained when Hb was added to [¹⁵N]Shr^HID2 (Fig. 2, compare a and c with b and d), indicating that both DUF1533 domains interact with Hb. In contrast, adding Hb to the [¹⁵N]Shr^L domain causes only minimal changes in the NMR spectrum indicating that the proteins do not interact (Fig. 2f).

Having established that DUF1533 domains bind to Hb, we next measured their affinities using ITC (Fig. 3, a–d, and Table 1). These data indicate that Shr^HID2 binds Hb with a K_D = 16.0 ± 3.4 μm in a process that is enthalpically favorable (Fig. 3b and Table 1). Shr^HID1 also binds Hb, but the interaction is weaker (K_D = ∼140 μm). The Shr^HID1–2 polypeptide containing both DUF1533 domains binds Hb with a K_D = 5.1 ± 0.4 μm (Fig. 3a and Table 1). Considering that the binding parameters for Shr^HID1–2 are distinct from either HID domain in isolation, it seems likely that Shr^HID1–2 exhibits binding avidity. If HID1 and HID2 were binding completely independently, one would expect the Shr^HID1–2–Hb binding parameters to more closely resemble the higher affinity Shr^HID2–Hb interaction. From these data, we conclude that both of Shr's DUF1533 domains bind Hb, and the second domain (Shr^HID2) is the most important for binding. Based on these results, we propose that the DUF1533 domains within Shr be renamed hemoglobin interacting domains (HIDs).

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.

Table 1.

Thermodynamic parameters and affinity data for Hb binding

Errors represent the standard deviation of three replicates.

	KD	ΔH0	ΔS0	ΔG0a	nb
	μm	kcal/mol	cal/mol·K	kcal/mol
Hb + ShrHID1–2	5.1 ± 0.4	−13.2 ± 1.1	−19.9 ± 3.9	−7.2 ± 0.1	0.58 ± 0.03
Hb + ShrHID2	16.0 ± 3.4	−7.8 ± 1.0	−4.3 ± 3.6	−6.6 ± 0.1	1.06 ± 0.08
Hb + ShrΔHID2	20.8 ± 0.7	−9.2 ± 1.1	−9.5 ± 3.7	−6.4 ± 0.1	1.14 ± 0.14
Hb + ShrHID1	∼143	NDc	ND	ND	ND

^a Samples were in buffer containing 20 mm sodium phosphate, 150 mm NaCl, 450 mm sucrose, pH 7.5, 298 K.

^bn refers to the molar ratio protein/Hb.

^c ND refers to “not determined.”

A NMR experiment was performed to ascertain whether the HIDs (DUF1533 domains) bind to the same site on Hb. In this experiment, the ability of unlabeled Shr^HID2 to displace Hb-bound [¹⁵N]Shr^HID1 was determined. Initially, unlabeled Hb was titrated into a solution of [¹⁵N]Shr^HID1 such that the signals in its ¹H–¹⁵N HSQC spectrum were broadened beyond detection as a result of complex formation (Fig. 4b). Unlabeled Shr^HID2 was then titrated into the solution, and the ¹H–¹⁵N HSQC spectrum was recorded to determine whether it displaces [¹⁵N]Shr^HID1 from Hb. Adding unlabeled Shr^HID2 to the solution causes the signals from [¹⁵N]Shr^HID1 to reappear in the HSQC spectrum, meaning that Shr^HID2 outcompetes [¹⁵N]Shr^HID1 for its binding site(s) located on Hb. The data show a progressive reappearance of the signals from [¹⁵N]Shr^HID1, starting when a 2-fold molar equivalent of Shr^HID2 is added (Fig. 4c). When 4-fold Shr^HID2 is added, the spectrum of [¹⁵N]Shr^HID1 is almost completely recovered, yet the signal intensities are approximately one-third as intense as in the original HSQC (Fig. 4d). Importantly, the reappearance of the NMR signals is not caused by simply diluting the NMR sample, as the signals from [¹⁵N]Shr^HID1 remain broadened in a control experiment in which the [¹⁵N]Shr^HID1–Hb complex was diluted only with buffer. As unlabeled Shr^HID2 displaces [¹⁵N]Shr^HID1 from Hb, we conclude that the isolated domains can compete for the same site(s) on Hb. However, the fact that this resulted in only a partial recovery of signal could indicate that Shr^HID1 binds a second site on Hb and binds to Hb more promiscuously than Shr^HID2.

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.

Two Shr^HID1–2 proteins bind to tetrameric Hb

The binding stoichiometry of the Shr^HID1–2–Hb complex was determined using size-exclusion chromatography with inline multiangle light scattering (SEC-MALS). When Shr^HID1–2 is mixed with the Hb tetramer at a ratio of 2:1 (Shr^HID1–2 to Hb tetramer units), a complex at 101 kDa is observed (Fig. 3e). This is consistent with ∼1.8 Shr^HID1–2 proteins binding to one Hb tetramer (calculated as follows: (101 kDa_(complex) − 56 kDa_{(Hb alone)})/25 kDa_(Shr^HID1–2 _alone) = 1.8). SEC-MALS experiments using higher Shr^HID1–2/Hb ratios (4:1 and 6:1) were also performed and yielded similar results, indicating that Hb is saturated, and a maximum of two Shr^HID1–2 proteins bind to one Hb tetramer. Notably, the stoichiometry predicted by SEC-MALS is similar to that obtained from curve-fitting of the ITC data (Table 1); two Shr^HID1–2 proteins bind to one Hb tetramer.

Analytical ultracentrifugation (AUC) was used to determine the stoichiometry of the Shr^HID2–Hb complex. This method was used instead of SEC-MALS because the molecular weights of Hb and its complex with Shr^HID2 are too similar, making it difficult to resolve these species by column chromatography. Similar to Shr^HID1–2, Shr^HID2 is monomeric in solution (Fig. 3f); it has a weighted average molecular weight of 13.3 kDa (Table S1), which corresponds closely to its theoretical molecular weight of 14.4 kDa (58). To simplify the analysis of the AUC data, binding experiments employed Hb0.1, a stabilized tetrameric from of Hb in which the α-globin chains are part of a single polypeptide. When Shr^HID2 is added to Hb0.1 at a 180-fold molar excess (Hb tetramer units), its weighted average molecular mass is 97.4 kDa (Fig. 3g and Table S1), consistent with two Shr^HID2 domains binding to one Hb0.1 tetramer. Hb0.1 is saturated with Shr^HID2, because similar molecular weights are measured when Shr^HID2 is present at 60-fold molar excess. Interestingly, the binding stoichiometry obtained from fitting the ITC data suggests that four Shr^HID2 proteins can bind to a tetramer, which differs from the 2:1 stoichiometry determined by AUC (Table 1). The origin of this discrepancy is unclear, but it may result from the different conditions employed in each experiment.

HID is a structurally novel Hb-binding domain

To gain insight into the molecular basis of Hb binding, we determined the crystal structure of HID2, the second DUF1533 domain within Shr. To facilitate its crystallization, we first used NMR to delineate regions within the Shr^HID2 polypeptide that are structurally ordered. Triple resonance methods were applied to [U-¹³C,U-¹⁵N]Shr^HID2, enabling ∼94% of its backbone resonances to be assigned (Fig. 5a). A heteronuclear {¹H} ¹⁵N NOE experiment was then performed to identify regions within Shr^HID2 that are mobile and unstructured (Fig. 5b). The majority of residues within Shr^HID2 are structurally ordered in the absence of Hb, as indicated by heteronuclear NOE values >0.6 for backbone amide groups that span residues Ile-179–Gln-283. However, ∼20 residues located at the N terminus of Shr^HID2 are disordered, as they possess low magnitude heteronuclear NOEs. We therefore produced a truncated variant of Shr^HID2 for X-ray crystallography studies that removes its unstructured N-terminal residues Lys-155–Ala-174 yielding a new construct (Shr^ΔHID2, residues Asn-175–Gln-285). Importantly, Shr^ΔHID2 binds to Hb with similar affinity and stoichiometry as Shr^HID2 (Fig. 3c and Table 1), indicating that the deleted N-terminal residues are not important for function.

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.

The structure of Shr^ΔHID2 was determined at 1.5 Å resolution using single-wavelength anomalous diffraction methods applied to native and KI-soaked crystals. Two Shr^ΔHID2 domains are present in the asymmetric unit (Fig. 6a), forming a 2-fold symmetry-related dimer that buries 517 Ų of surface area (59). As SEC-MALS and AUC analyses indicate that Shr^ΔHID2 is monomeric, the dimer observed in the structure is likely an artifact caused by the high concentration of protein present in the crystal. The two polypeptide chains in the dimer possess nearly identical atomic structures, as their heavy atom coordinates can be superimposed with a r.m.s.d. of 0.80 Å. The polypeptides are well-defined by the electron density, with the exception of residue Asp-226 in chain A and residues Lys-284 and Gln-285 in chain B. Complete structural and data statistics are presented in Table 2.

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.

Table 2.

Structure statistics on Shr^ΔHID2

	Native	KI soak (SAD)
Data collection
Beamline	APS	UCLA-HTC
Wavelength (Å)	0.9793	1.5418
Space group	P212121	P212121
Cell dimensions
a, b, and c (Å)	32.47, 59.14, 102.71	31.51, 57.90, 102.00
α, β, γ (°)	90, 90, 90	90, 90, 90
Resolution (Å)	19.4–1.5 (1.554–1.5)a	19.4–1.818 (1.883–1.818)
Rmerge	0.04259 (0.5419)	0.0295 (0.08212)
Rmeas	0.04649 (0.5921)	0.03443 (0.1068)
Rpim	0.01836 (0.2356)	0.01761 (0.06735)
Mean I/σ(I)	23.21 (3.34)	28.78 (8.12)
Completeness (%)	99.75 (99.63)	99.66 (99.61)
No. of total reflections	206,028 (19825)	114,307 (3963)
No. of unique reflections	32,457 (3194)	32,554 (1808)
Multiplicity	6.3 (6.2)	3.5 (2.0)
Wilson B-factor	19.84	18.98
CC1/2	0.999 (0.914)	0.999 (0.985)
CC*	1 (0.977)	1 (0.996)
Refinement
Resolution (Å)	19.7–1.5 (1.55–1.5)
No. of reflections	32,457
Rwork/Rfree	0.197/0.222 (0.2388/0.2685)
No. of atoms	1824
Protein	1696
Sulfate	15
Water	113
Average B-factors
Protein	25.22
Sulfate	54.15
Water	29.91
r.m.s.d.
Bond lengths (Å)	0.010
Bond angles (°)	1.17
Ramachandran plot
Favored (%)	95.35
Allowed (%)	4.65
Outliers (%)	0.00
PDB code	6DKQ

^a Statistics for the highest resolution shell are shown in parentheses.

Shr^ΔHID2 adopts a β-sandwich–type fold in which two antiparallel β-sheets are flanked by an α-helix. One face of the sandwich is formed by a sheet containing strands β1, β2, β6, and β5 (β-sheet 1), whereas the other face is formed by a sheet containing strands β9, β8, β7, β3, and β4 (β-sheet 2) (Fig. 6b). Strands β1 and β2 form an antiparallel β-hairpin. The β2 strand is then followed by a long loop that is connected to a two-turn α-helix that is positioned nearly perpendicular with respect to β-sheet 2. Following the helix, strand β3 makes antiparallel contacts with strand β7 in β-sheet 2. Strand β4 then forms part of a β-hairpin with β3 before being followed by an extended loop that winds around and extends over the top of β-sheet 1. This is followed by strand β5 that forms a β-hairpin with strand β6 and together with strands β1 and β2 comprise β-sheet 1. The structure is completed by a β-meander that is constructed from strands β7, β8, and β9 within β-sheet 2. Shr^HID1 presumably adopts a similar structure, as it shares 27% sequence identity with Shr^HID2. The solvent-exposed side of β-sheet 1 is predominantly acidic, whereas on β-sheet 2 there is a greater balance of acidic and basic residues (Fig. 6c). It is worth mentioning that the HID domain is larger than the predicted DUF1533 domain, wherein β1, β2, and the loop connecting it to α1 as well as β9 are all located outside of the predicated DUF1533 domain in the primary sequence.

The HID is a structurally unique bacterial Hb-binding domain. Many species of Gram-positive bacteria that are related to S. pyogenes employ NEAT domains to bind Hb. Based on a Dali analysis, the Shr^ΔHID2 and NEAT domains adopt distinct structures; their Z-score does not exceed the threshold for a strong match as defined by Holm et al. (60). Moreover, Shr^ΔHID2 and NEAT domains have distinct secondary structure topologies, and Shr^ΔHID2 lacks the conserved aromatic motif that is present in Hb-binding NEAT domains (Fig. 7, a and b). Shr^ΔHID2 is also structurally distinct from Hb receptors found in other human pathogens, such as HpuA (61), ShuA (62), and HpHbR (63). The closest HID structural homolog in the Protein Data Bank is the CBM46 domain present in the Cel5B cellulase from Bacillus halodurans (PDB code 4uz8); the proteins can be superimposed with an r.m.s.d. of 2.1 Å and have a Dali Z-score of 7.9, which is deemed significant (60). However, the proteins have distinct functions, as key residues in CBM46 that mediate carbohydrate binding are poorly conserved in Shr^ΔHID2.

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.

HIDs within Shr^HID1 and Shr^HID2 engage Hb using a conserved tyrosine residue

We attempted to identify surface residues in the HID that mediate Hb binding by aligning the primary sequences of the DUF1533 domains and their flanking regions from putative Hb receptors. The primary sequences of DUF1533 domains from four Shr orthologues present in Clostridium novyi, Streptococcus iniae, Streptococcus equi, and Streptococcus dysgalactiae were aligned to Shr^HID1 and Shr^HID2 (Fig. S2). Six residues are universally conserved, of which only one has a side chain that is completely surface-exposed, Tyr-197 (Shr^HID2 labeling). A Y197A mutation was introduced into Shr^HID2 (Shr^HID2-Y197A), and its ability to bind Hb was determined using the aforementioned NMR titration experiment. In marked contrast to WT Shr^HID2, adding Hb to [¹⁵N]Shr^HID2-Y197A does not cause its NMR signals to broaden significantly, even when Hb is in excess (2:1 Hb/Shr^HID2-Y197A) (Fig. 8a). As the Y197A mutation does not disrupt the structure of Shr^HID2-Y197A, the side chain of Tyr-197 presumably forms contacts with Hb that are required for binding. The corresponding tyrosine mutation was also introduced into Shr^HID1 (Y55A, Shr^HID1-Y55A) and was also shown to disrupt Hb binding (Fig. 8b). We conclude that a conserved tyrosine residue in Shr^HID1 and Shr^HID2 is critical for Hb binding and that the domains likely engage Hb in a similar manner.

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.

Discussion

Human hemoglobin is a rich source of iron that bacterial pathogens capture using surface-displayed and secreted Hb receptors (64). In Gram-positive bacteria, these receptors typically interact with Hb via NEAT domains (23). Interestingly, the Hb receptor in S. pyogenes, called Shr, appears to bind Hb via a novel mechanism. Its primary sequence has been divided into two regions: NTR that binds Hb, and CTR that binds hemin (8, 11). Intriguingly, based on its primary sequence, the NTR does not contain a NEAT domain suggesting that it engages Hb in a unique manner. To learn how Shr interacts with Hb, we first used NMR spectroscopy to delineate residues within its NTR that form autonomously folded domains. Three regions within the NTR are structured, two modules previously defined as domains of unknown function (DUF1533 domains) and a third module located near the C terminus of the NTR that is predicted to contain several α-helices (Fig. 1). NMR titration experiments conclusively demonstrate that only the DUF1533 domains within the NTR interact with Hb (Fig. 2). They therefore represent previously uncharacterized HIDs. In Shr they are HID1 (residues 26–148) and HID2 (residues 155–285). Interactions originating from HID2 contribute significantly to Shr's Hb binding affinity, because based on ITC measurements it binds to Hb with a K_D = 16.0 ± 3.4 μm, whereas the isolated HID1 domain has significantly weaker affinity (K_D = ∼140 μm). Studies of a polypeptide containing both domains (Shr^HID1–2), suggest avid binding to Hb. The 3-fold decrease in K_D (5.1 ± 0.4 μm) afforded by HID1 in the Shr^HID1–2 construct would seem to indicate that binding of one HID domain facilitates binding of the other resulting in the higher apparent affinity observed for the Shr^HID1–2–Hb interaction. Tsumoto and co-workers (11) have measured the binding affinity of residues Gln-22–Val-364 of Shr, which contains both HIDs and the linker domain within the NTR. This polypeptide binds to Hb with similar affinity as Shr^HID1–2 (K_D = 6.7 μm), further demonstrating that only the HIDs within the NTR mediate Hb binding.

The crystal structure of HID2 reveals that it adopts a compact β-sandwich type fold that is distinct from previously characterized Hb-binding proteins (Fig. 6b). Structures of three different NEAT domains in complex with Hb have been determined, including the first and second NEAT domains from the S. aureus IsdH receptor (IsdH^N1 and IsdH^N2) and the first NEAT domain from the S. aureus IsdB protein (IsdB^N1) (44, 50, 65). These NEAT domains share significant primary sequence homology with one another and engage Hb in a similar manner. The globin chain in Hb is contacted by residues in the NEAT domains that are located in an α-helix that is positioned between strands β1b and β2, and by residues located within nearby loops (loops connecting strands β3–β4 and β5b–β6). Each NEAT domain contains a conserved aromatic signature sequence ((F/Y)YH(F/Y)) that forms the Hb contacting α-helix that when mutated disrupts binding. The HIDs within Shr must bind to Hb in a fundamentally distinct manner, as their tertiary structures differ substantially from NEAT domains. Moreover, HID primary sequences do not contain the Hb-contacting aromatic motif. Atomic structures of other Hb receptors from eukaryotic and Gram-negative microbes have also been determined and do not share significant structural homology with the HID.

To identify the surface on HIDs that contact Hb, we compared their primary sequences and performed targeted mutagenesis. Six residues are completely conserved, but only Tyr-197 (HID2 numbering) is fully solvent-exposed in the structure of HID2 and conserved. Tyr-197 is located in the extended loop that precedes the α-helix and is surrounded by other less well-conserved residues that may mediate Hb interactions. Mutational exchange of the tyrosine to alanine in either HID1 or HID2 abrogates Hb binding, substantiating its functional importance (Fig. 8). The HID was originally named DUF1533. At present, more than 150 proteins containing DUF1533 domains from 71 distinct species of bacteria are known. Notably, these conserved tyrosine residues lie outside the predicted DUF1533 domains in the N-terminal regions of the HIDs. It is therefore unclear whether all of these sequence-related domains will also bind Hb, as some lack the key tyrosine residue and they are present in proteins that are unlikely to be orthologs of Shr because they contain other domains that are predicted to have enzymatic functions.

In vitro binding studies indicate that a single Hb tetramer is engaged by two Shr receptors. SEC-MALS experiments demonstrate that a Shr^HID1–2 polypeptide containing both HID1 and HID2 forms a complex with the Hb tetramer that has 2:1 stoichiometry (Fig. 3e). It is likely that the intact Shr protein binds with similar stoichiometry, as residues outside the NTR do not mediate interactions with Hb (8). Within the 2:1 Shr^HID1–2–Hb complex, we speculate that HID1 and HID2 domains likely engage distinct sites on the Hb tetramer. In this scenario, each of the domains would bind a globin subunit, with interactions originating from HID2 being the most important. This model is supported by the stoichiometry obtained for the Shr^HID1–2–Hb interaction by SEC-MALS. It is also consistent with the results of the NMR experiments that showed that the isolated domains competitively bind Hb if it is assumed that the high-protein concentrations used in the NMR studies caused the domains to promiscuously bind to the globin subunits and/or the domains bind differently to Hb in isolation as compared with Shr^HID1–2. Furthermore, the mutagenesis studies in combination with the NMR competition experiments suggest that both HID's engage Hb via the same interface. Taken together, this leads us to conclude that each globin chain likely contains a single HID-binding site. It is interesting to note that HID binding to Hb is entropically unfavorable for both the isolated domains and Shr^HID1–2 (Table 1). It is possible that residues within the domain(s) undergo a disorder to order transition upon binding Hb, as is the case for the Hb-binding NEAT domains within the IsdH and IsdB S. aureus Hb receptors (50, 65). However, the origin of this effect remains unclear, as the heteronuclear NOE data of apo-HID2 do not reveal the presence of large disordered loops. Structures of the Shr–:Hb complex are required to understand the molecular origins of these energetic effects.

Based on the domain architecture that we have elucidated, Shr may use a tri-domain unit to extract hemin from Hb. Only the heme extraction mechanisms used by the S. aureus IsdB and IsdH proteins are well-understood. These proteins share similar primary sequences and extract hemin from Hb using a tri-domain unit, in which a helical domain separates N- and C-terminal NEAT domains that bind to Hb and hemin, respectively (42). Detailed studies of IsdH indicate that its N-terminal NEAT domain (N2 domain) engages the A-helix within a globin chain contributing ∼95% of the total binding standard free energy (43). This positions the remaining helical and NEAT domains near the hemin pocket on the same globin subunit, thereby enabling them to distort Hb's F-helix to trigger hemin release. IsdB also contains a homologous tri-domain unit that binds to Hb in a similar manner (50). Interestingly, Shr contains a hemin- binding NEAT domain (NEAT1) immediately following the HID2 and linker domains we have identified (Fig. 1). Thus, residues 175–502 in Shr form a D2–linker–NEAT1 tri-domain segment that is similar to those present in IsdB and IsdH. It is possible that Shr uses a similar mechanism as IsdH/B to accelerate hemin release from Hb in which HID2 binds to a globin chain to “deliver” the NEAT1 domain near Hb's hemin pocket. Interestingly, both the Shr and IsdH receptors supplement their tri-domain segments with an additional N-terminal Hb-binding domain (HID1 in Shr and the N1 NEAT domain in IsdH). Recent studies indicate that the supplemental N1 domain in IsdH may function to slow the rate of Hb removal from the blood by obstructing interactions with the macrophage-specific endocytic receptor CD163, thereby advantageously prolonging microbial access to Hb's heme iron (66). Additional studies will be needed to determine whether the HID1 domain in Shr performs a similar function.

Collectively, the data presented in this paper provide new insight into how Shr captures Hb on the cell surface. S. pyogenes presumably encounters the dimeric form of Hb, as rupture of the red blood cells is expected to dramatically dilute Hb causing the tetramer to dissociate in αβ dimers. As we have shown that two receptors bind a Hb tetramer, on the cell surface Shr likely engages the Hb αβ dimer with 1:1 stoichiometry, such that its HID1 and HID2 domains each engage a globin subunit. Based on previous hemin transfer studies between Shr and Hb (8, 9), it seems likely that Hb's hemin molecule is transferred to Shr's NEAT1 domain, which is proximally positioned to HID2. The domain architecture of Shr^{HID2–L–N1} is similar to IsdH^N2N3, a Hb- and a hemin-binding domain separated by an α-helical linker. We have previously shown that IsdH^N2N3 accelerates hemin release from Hb by interactions with the linker domain, which distorts the Hb's F-helix (45, 46). The similar domain architecture between IsdH^N2N3 and Shr suggests that the linker domain in Shr may also facilitate hemin release after it engages Hb. Further insight into the mechanism of Hb capture will be gained by structural studies on the Shr–Hb complex.

Experimental procedures

Cloning, protein expression, and purification

Standard methods were used to construct expression plasmids that produced the following polypeptides: Shr^HID1 (residues 26–148); Shr^HID2 (residues 155–285); Shr^ΔHID2 (residues 175–285); Shr^HID1–2 (residues 26–285); and Shr^L (residues 294–364). Proteins were expressed from pSUMO-based vectors (LifeSensors) that were transformed into Escherichia coli BL21(DE3) cells (New England Biolabs, Beverly, MA). Cultures were grown at 37 °C to an A₆₀₀ of 0.6–0.8 before induction with isopropyl β-d-thiogalactoside at a final concentration of 1 mm. Induction proceeded at 25 °C overnight before harvesting the cells by centrifugation. The cell pellet was then resuspended in lysis buffer containing: 50 mm NaH₂PO₄/Na₂HPO₄, pH 7.0, 300 mm NaCl. A protease inhibitor mixture (Calbiochem) and phenylmethylsulfonyl fluoride (Sigma) was also added to the lysis buffer. Cells were lysed by sonication, and the lysate was clarified by centrifugation (27,200 × g). The lysate was then loaded onto a Co²⁺-chelating column (ThermoFisher Scientific, Waltham, MA), and unbound proteins were removed by washing the column with lysis buffer (5 column volume (CV) equivalents). Nonspecifically bound proteins were also removed by applying 5 CVs of wash buffer (lysis buffer + 10 mm imidazole) and 5 CVs of lysis buffer. The N-terminal His₆–SUMO tag was then cleaved with the ULP1 protease and eluted by washing the column with lysis buffer. If necessary, proteins were purified using an SEC column as described by the vendor (Sephacryl, GE Healthcare). Human Hb was prepared as described previously from the blood of a healthy donor provided by the CFAR Virology Core Lab at the UCLA AIDS Institute (43). Purified Hb0.1 was a generous gift of the Olson laboratory.

NMR spectroscopy

Uniformly ¹³C- and ¹⁵N-labeled Shr^HID2 was expressed in E. coli BL21(DE3) cells grown in M9 medium supplemented with ¹⁵NH₄Cl and [¹³C₆]glucose. The NMR sample of Shr^HID2 contained 1.1 mm [U-¹⁵N,U-¹³C]Shr(155–285) dissolved in 50 mm NaH₂PO₄/Na₂HPO₄, pH 6.0, 50 mm NaCl, 0.01% NaN₃ with 92:8% H₂O/D₂O. NMR experiments were performed at 298 K on Bruker DRX 500, DRX 600 MHz, and AVANCE 800 MHz spectrometers equipped with triple resonance cryogenic probes. NMR spectra were processed using NMRPipe (67) and TopSpin3 (68) and analyzed using CARA (69). Backbone ¹H, ¹³C, and ¹⁵N chemical shifts were assigned by analyzing data from the following experiments: 2D ¹H-¹⁵N HSQC, 2D ¹H-¹³C HSQC, 3D CBCA(CO)NH, HNCACB, HNCO, HN(CA)CO, HNCA, HNHA, HNHB, HBHA(CO)NH, HBHANH, and CC(CO)NH (Fig. 5a). Assignments have been deposited to the BMRB under accession number 27550. Heteronuclear {¹H} ¹⁵N NOE experiments were collected in an interleaved manner and analyzed using Sparky (70). The average and standard deviation of three replicates are reported (Fig. 5b).

NMR Hb titration experiments employed 200 μm uniformly ¹⁵N-labeled Shr constructs that were dissolved in 50 mm NaH₂PO₄/Na₂HPO₄, pH 6.8, 100 mm NaCl, 92% H₂O/8% D₂O. The Hb titrant was dissolved in 50 mm NaH₂PO₄/Na₂HPO₄, pH 6.8, 100 mm NaCl and added from a 3.5–5.5 mm stock solution (heme units). Hb was added to the sample at a 1:1 ratio of tetramer to ¹⁵N-labeled Shr construct. The NMR competition experiments were carried using conditions that were similar to those used for the Hb titration experiments. A sample of uniformly ¹⁵N-labeled Shr^HID1 dissolved in 50 mm NaH₂PO₄/Na₂HPO₄, pH 6.8, 100 mm NaCl, 92% H₂O/8% D₂O to a final concentration of 200 μm was prepared. Buffer-matched Hb in its carbonmonoxy state was then added such that the final ratio was 1:2, [¹⁵N]Shr^HID1/Hb. Unlabeled Shr^HID2 was then added to achieve a ratio of [¹⁵N]Shr^HID1/Hb/Shr^HID2 = 1:2:2 or 1:2:4. All samples were incubated for 30 min at room temperature prior to data acquisition. For each ¹H–¹⁵N HSQC experiment, the number of scans was adjusted to compensate for changes in protein concentration.

Isothermal titration calorimetry (ITC)

ITC measurements were performed using a MicroCal iTC200 calorimeter (GE Healthcare) at 25 °C. The Hb (in the carbonmonoxy state) and Shr constructs were buffer-matched in 20 mm NaH₂PO/Na₂HPO₄, pH 7.5, 150 mm NaCl, 450 mm sucrose. The cell was filled with 25–35 μm Hb (concentration reported on a heme/globin chain basis), and the syringe was filled with either 700 μm Shr^HID1, 500 μm Shr^HID2, 550–600 μm Shr^ΔHID2, or 350–500 μm Shr^HID1–2. Twenty injections were performed using 2.0-μl injection volumes at 180-s intervals. For each of the Shr constructs, an experiment was carried out to control for the heats of dilution in which the respective Shr construct was titrated into buffer, and the control data were subtracted from the experimental data. ORIGIN software was used to fit the data to a single-site binding model.

SEC-MALS

The analytical size-exclusion column WTC-030S5 (Wyatt Technology) was equilibrated in 20 mm NaH₂PO/Na₂HPO₄, pH 7.5, 150 mm NaCl using an AKTA pure (GE Healthcare). For each protein or complex, a 100-μl (buffer-matched) sample was loaded: 1.2 mm HbCO (heme basis), 600 μm Shr^HID1–2, and the complex of HbCO at 1.2 mm and Shr^HID1–2 at 600 μm, 1.2 mm, and 1.8 mm. During elution, light scattering was measured with a miniDAWN TREOS (Wyatt Technology), and the refractive index (n) was measured with an Optilab T-rEX system (Wyatt Technology). Data were analyzed using ASTRA software (version 6.1) to obtain average molecular weights. The dn/dc value (where c is the concentration) for the calculation was set to 0.185 ml/g for Hb (66). Based on the primary sequence, the dn/dc values for Shr^HID1–2 and Shr^ΔHID2 were calculated to be 0.187 and 0.188 ml/g, respectively. For the complex of Shr^HID1–2 with Hb, the dn/dc value was calculated to be 0.186 ml/g based on the equation for the weight average sum. Theoretical molecular weights were obtained using the ProtParam server based on the primary amino acid sequence (58).

Analytical ultracentrifugation (AUC)

Sedimentation equilibrium experiments were performed at 20 °C using a Beckman Optima XL-A analytical ultracentrifuge equipped with an An60-Ti rotor. Native HbA has a dimer/tetramer equilibrium, which complicates the determination of the Shr^HID2–Hb binding stoichiometry by AUC. To simplify the analysis, a mutant variety of Hb, which stabilizes the tetrameric form, was employed (Hb0.1) (71, 72). Absorption optics at 280 nm were carried out for Shr^HID2 alone and at 412 nm for Hb0.1 alone or in complex with Shr^HID2 so that only the heme molecule was detected. All experiments were carried out in 20 mm NaH₂PO/Na₂HPO₄, pH 7.5, 150 mm NaCl. 450 mm sucrose. To prevent heme release, Hb0.1 in the carbon monoxide state was used. Three mm pathlength double sector cells were used for all samples and were purged with CO before sealing the cells to prevent oxidation. Sedimentation equilibrium profiles were measured at 8,000 (∼5,200 × g), 11,000 (∼9,700 × g), and 26,000 rpm (∼54,500 × g) for Shr^HID2 alone, with the last being used for the analysis. For Hb0.1 alone and in complex with Shr^HID2, sedimentation equilibrium profiles were measured at 11,000 (∼9,700 × g) and 13,000 rpm (∼13,600 × g), with the latter being used for the analysis. A third measurement was made at 30,000 rpm (∼72,500 × g) and was used to determine the baseline. Samples of Hb0.1 and Shr^HID2 alone contained 5.5 and 248 μm protein, respectively. For data collected at a 15:1 ratio of Shr^D2/Hb0.1, the sample contained 6.2 and 89 μm Hb0.1 and Shr^HID2, respectively. For the 45:1 ratio of Shr^HID2/Hb0.1, the sample contained 5.5 and 248 μm Hb0.1 and Shr^HID2, respectively. Weight average molecular masses were determined by fitting with a nonlinear least-squares exponential fit for a single ideal species using Beckman Origin-based software (version 3.01). Partial specific volumes were calculated from the amino acid compositions and corrected to 20 °C. They were 0.740 for Shr^HID2, 0.749 for Hb0.1, and 0.746 for the complex.

X-ray crystallography

Crystals of Shr^ΔHID2 were produced from a stock of 52 mg/ml (4.2 mm) dissolved in 20 mm NaH₂PO₄/Na₂HPO₄, pH 6.5. Crystals were grown using the hanging drop, vapor diffusion method against a mother liquor of 0.1 m sodium acetate, pH 4.5, 0.1–0.2 m lithium sulfate, 30–50% (w/v) PEG 400. Crystals developed after ∼24 h at room temperature. Data sets were collected on two crystals: Shr^ΔHID2 (native) and Shr^ΔHID2 soaked for 30 s in mother liquor containing 35.9% (w/v) PEG 400 and 460 mm KI (Shr^ΔHID2 + KI) (73). The KI dataset was collected on a Rigaku FRE+ generator with a copper anode equipped with a Rigaku HTC image plate detector (λ = 1.5418 Å). The Matthews coefficient indicated that there were two Shr^ΔHID2 molecules in the asymmetric unit using the online server MATTPROB (74). XDS/XSCALE was used to index, integrate, and scale the data (75). Conservative resolution limits were applied to balance the calculated I/σ, R_sym, and CC1/2 in the highest resolution shell. The structure of Shr^ΔHID2 was solved by single-wavelength anomalous dispersion on Shr^ΔHID2 + KI using HKL2MAP (76), the graphical user interface for the SHELXC/D/E programs. Four iodide sites were found by SHELXD (77). SHELXE (78) was used to assign the hand, produce the first set of phases, and perform solvent flattening. The final figure of merit was 0.767. Further density modification and automatic model building was performed by SHARP (79). SHARP built residues 2–111 of chain A and 8–109 of chain B in the initial model out of 112 residues in the construct. The Shr^ΔHID2 + KI model was refined to 1.8 Å. This model was further refined against the high-resolution 1.5 Å native data set obtained at the Advanced Photon Source on beamline 24-ID-C on a DECTRIS-PILATUS 6M detector (λ = 0.9793 Å). Initial rounds of structure refinement were carried out using Coot (80) and PHENIX (81). Later rounds of refinement were carried out using Coot (80) and BUSTER (82) with TLS refinement (83). Structure statistics are presented in Table 2. The coordinates of the final model and structure factors have been deposited in the Protein Data Bank under PDB code 6DKQ.

Author contributions

R. M. and R. T. C. conceptualization; R. M., D. C., and M. L. P. data curation; R. M., D. C., and M. L. P. formal analysis; R. M. investigation; R. M. and R. T. C. writing-original draft; D. C., M. J. C., and M. L. P. resources; D. C. and M. L. P. software; D. C. and M. L. P. validation; D. C. methodology; R. T. C. supervision; R. T. C. funding acquisition; R. T. C. project administration.