Structure and role of the linker domain of the iron surface-determinant protein IsdH in heme transportation in Staphylococcus aureus

Abstract

Staphylococcus aureus is a major cause of deadly nosocomial infections, a severe problem fueled by the steady increase of resistant bacteria. The iron surface determinant (Isd) system is a family of proteins that acquire nutritional iron from the host organism, helping the bacterium to proliferate during infection, and therefore represents a promising antibacterial target. In particular, the surface protein IsdH captures hemoglobin (Hb) and acquires the heme moiety containing the iron atom. Structurally, IsdH comprises three distinctive NEAr-iron Transporter (NEAT) domains connected by linker domains. The objective of this study was to characterize the linker region between NEAT2 and NEAT3 from various biophysical viewpoints and thereby advance our understanding of its role in the molecular mechanism of heme extraction. We demonstrate the linker region contributes to the stability of the bound protein, likely influencing the flexibility and orientation of the NEAT3 domain in its interaction with Hb, but only exerts a modest contribution to the affinity of IsdH for heme. Based on these data, we suggest that the flexible nature of the linker facilitates the precise positioning of NEAT3 to acquire heme. In addition, we also found that residues His45 and His89 of Hb located in the heme transfer route toward IsdH do not play a critical role in the transfer rate-determining step. In conclusion, this study clarifies key elements of the mechanism of heme extraction of human Hb by IsdH, providing key insights into the Isd system and other protein systems containing NEAT domains.

Keywords: Staphylococcus aureus; heme; heme acquisition; heme transport; hemoglobin; hemoglobin receptor; hydrogen exchange mass spectrometry; iron surface determinant system; structure-function; x-ray crystallography.

Figure 1

Crystal structure of IsdH linker-NEAT3 in complex with Fe(III)-PPIX.A, modular structure of IsdH. B, overall structure of linker-NEAT3 at 1.8 Å resolution. The linker and NEAT3 domains are shown in green and blue, respectively. The heme group is depicted with orange sticks. The iron atom is shown as an orange sphere. Key residues for binding heme, Tyr642 and Tyr646, are also depicted with blue sticks. The identity of the first and the last residue of each domain is shown. C, close-up view of the heme-binding pocket in NEAT3. Some residues surrounding heme are depicted with sticks. D, surface representation using the same color scheme as above. E, close-up view of the interaction area between linker and NEAT3 domains. Relevant residues belonging to linker and NEAT3 domains are depicted with green and blue sticks, respectively. The figure was prepared using UCSF Chimera (53). NEAT, NEAr-iron Transporter.

Figure 2

Mapping flexibility of linker-IsdH by HDX-MS.A, four segments of interest are highlighted on the structure of IsdH linker-NEAT3 in complex with Fe(III)-PPIX. The flexible and/or exposed areas are highlighted, in green for the linker domain and in blue for the NEAT3 domain. The heme moiety is showed in orange. B, time course of deuterium exchange for the four regions mapped above. Each panel shows the degree of deuterium exchange for each segment (indicated at the top of each panel) in the heme-free (black line) and heme-bound (red line) forms. HDX-MS, hydrogen-deuterium exchange mass spectrometry; NEAT, NEAr-iron Transporter.

Figure 3

Thermostability of IsdH constructs.A and B, DSC thermograms of (A) NEAT3 and (B) linker-NEAT3. Data for heme-free and heme-bound forms are shown in blue and red traces, respectively. The black and gray traces correspond to the overall fitting and the single-peak fittings (only in the sample of linker-NEAT3 without heme), respectively. Scans between 20 and 90 °C were performed in PBS at a protein concentration of 100 μM. DSC, differential scanning calorimetry; NEAT, NEAr-iron Transporter.

Figure 4

Binding of heme to IsdH determined by ITC.A, binding of heme to NEAT3. B, binding of heme to linker-NEAT3. The top panels correspond to the titration kinetics, whereas the bottom panels represent the integrated binding isotherms. The experiments were carried out in PBS at pH 7.4 supplemented with 5% DMSO at a concentration of protein and heme of 4 and 50 μM, respectively. Molar ratio refers to the relative concentration (molar ratio) of heme with respect to protein throughout the titration. The binding enthalpy (ΔH) and the dissociation constant (K_D) were determined by nonlinear regression of the integrated data to a one-site binding model with the program Origin. The values of k_on and k_off were determined from the titration kinetics (top portion of each panel) with the software AFFINImeter (50). All parameters are given in Table 2. DMSO, dimethyl sulfoxide; ITC, isothermal titration calorimetry; NEAT, NEAr-iron Transporter.

Figure 5

Heme transfer from linker-NEAT3 to NEAT3.A and B, absorbance spectra of IsdH constructs (A) before the transfer and (B) after the transfer. The spectra of NEAT3 and linker-NEAT3 are shown with black and red traces, respectively. Absorbance was normalized to acquire a comparable spectrum of the Soret region (heme region). Details of the experiment are described in Experimental procedures. NEAT, NEAr-iron Transporter.

Figure 6

Comparison of structures and heme movement.A, superposition of the crystal structure of human Hb (PDB entry code 3P5Q) (54), human Hb in complex with NEAT2-linker-NEAT3^Y642A (PDB entry code 4XS0) (19), and linker-NEAT3 in complex with heme (this work). Human Hb without IsdH bound is represented with brown ribbons, whereas human Hb in complex with IsdH is shown in pink. The NEAT2 domain is not shown. The linker and NEAT3^Y642A domains of the protein bound to Hb are shown in dark green and dark blue, respectively. Linker and NEAT3 in complex with heme are depicted in light green and light blue, respectively. The heme moiety bound to Hb in the structure in complex with IsdH is depicted in pink. The heme moiety bound to linker-NEAT3 is depicted with orange sticks. The iron atom is depicted with a sphere. Some key residues and the heme moieties are labeled. B, close-up view of the heme-binding region showing the movement of heme (indicated by the dashed arrow) to reach the binding pocket of IsdH. C, close-up view of the rotation of heme necessary to accommodate within the binding pocket of IsdH. The figure was prepared with UCSF Chimera (53). NEAT, NEAr-iron Transporter.

Figure 7

Time course of heme acquisition from Hb.A, normalized UV/Vis absorbance at 405 nm collected over 20 s after mixing of different samples of rHb and heme-free NEAT1-NEAT3 in PBS with 0.5 M sucrose. The colored dotted plots corresponded to the normalized mean of absorbance of four independent experiments. The data colored in red, blue, gray, and green correspond to the values obtained for rHb WT, the single mutant H45A, the single mutant H89A, and the double mutant H45A/H89A, respectively. The black traces represent the fitting using a two-phase decay function. B, representation of k_fast. C, representation of k_slow. The middle line in each box represents the average of the independent values, which are represented by the dots. The upper and lower error bars represent the values of the standard deviation normalization and fitting were performed using the GraphPad Prism software. Other experimental details are as described in Experimental procedures. NEAT, NEAr-iron Transporter.

Figure 8

Model of heme acquisition of IsdH from Hb. Hb is represented with a pink circle, heme with a red star, NEAT3 with an indented blue circle, and the linker with three green rectangles. For clarity purposes the domain NEAT1-2 responsible for binding to Hb is not shown. A, Hb and linker-NEAT3 before binding. The F-helix of hemoglobin is represented as a safety pin and the linker flexibility indicated with the open padlock and the multiple conformations. B, upon binding, the linker and NEAT3 destabilize the F-helix of Hb (represented by the safety pin in the open conformation), which weakens heme binding to Hb and facilitates its movement to IsdH NEAT3. The loss of flexibility of the linker was represented with the locked padlock and a single conformation of the linker. C, after heme is acquired, IsdH releases the Hb moiety. The linker in the heme-bound form remains in a locked conformation. D, IsdH with heme bound interacts with IsdC or IsdA, and heme is transferred. E, as a result of heme transfer to IsdC or IsdA, the linker recovers its flexibility and gets ready to bind another molecule of Hb. NEAT, NEAr-iron Transporter.