Mapping ultra-weak protein-protein interactions between heme transporters of Staphylococcus aureus

Abstract

Iron is an essential nutrient for the proliferation of Staphylococcus aureus during bacterial infections. The iron-regulated surface determinant (Isd) system of S. aureus transports and metabolizes iron porphyrin (heme) captured from the host organism. Transportation of heme across the thick cell wall of this bacterium requires multiple relay points. The mechanism by which heme is physically transferred between Isd transporters is largely unknown because of the transient nature of the interactions involved. Herein, we show that the IsdC transporter not only passes heme ligand to another class of Isd transporter, as previously known, but can also perform self-transfer reactions. IsdA shows a similar ability. A genetically encoded photoreactive probe was used to survey the regions of IsdC involved in self-dimerization. We propose an updated model that explicitly considers self-transfer reactions to explain heme delivery across the cell wall. An analogous photo-cross-linking strategy was employed to map transient interactions between IsdC and IsdE transporters. These experiments identified a key structural element involved in the rapid and specific transfer of heme from IsdC to IsdE. The resulting structural model was validated with a chimeric version of the homologous transporter IsdA. Overall, our results show that the ultra-weak interactions between Isd transporters are governed by bona fide protein structural motifs.

FIGURE 1.

Self-transfer of heme between Isd transporters. A, experimental design. Untagged holoprotein and His₆-tagged apoprotein were mixed. After a brief incubation step, the tagged and untagged proteins were separated by IMAC. Hm, heme iron. Shown are spectra of holo-IsdC (B), His₆-apo-IsdC (C), holo-IsdA (D), and His₆-apo-IsdA (E) before (dashed lines) and after (solid lines) a 10-min incubation, followed by IMAC. Holo-IsdC and holo-IsdA were mixed with His₆-apo-IsdC and His₆-apo-IsdA, respectively. Apoprotein was added in excess to a solution containing 3 μm holo-form. In the spectra, the downward-pointing arrows indicate heme depletion, and the upward-pointing arrows indicate heme acquisition.

FIGURE 2.

Self-dimerization of IsdC detected by photo-cross-linking. A, identification of the photo-cross-linked self-dimer of IsdC mutein I48X by immunoblotting with an anti-His probe. Samples were irradiated with UV light (365 nm) for 30 min. The arrow indicates the position of the photo-cross-linked product in the Western blot. B, interaction analysis with 17 different muteins. Only the 40–50-kDa region of the Western blot is shown. C, interaction map. Mutated residues leading to photo-cross-linked products are depicted in green. Residues in orange indicate no dimerization reaction. Heme is shown in dark blue. The magenta sphere corresponds to iron. The majority of residues involved in dimerization are clustered around the distal 3₁₀-helix and the β7/β8 strand. Molecular graphics images were produced with the UCSF Chimera package (54) using the coordinates of holo-IsdC (Protein Data Bank code 2O6P) (15).

FIGURE 3.

Transient interactions between IsdC and IsdE detected by photo-cross-linking. A, photo-cross-linking experiment between IsdC mutein F130X and wild-type IsdE. The arrow indicates the position of the cross-linked IsdC-IsdE heterodimer. B, detection of a photo-cross-linked product between wild-type IsdC and either IsdE mutein D101X or IsdE mutein Y115X. C, interaction analysis. The 50–60-kDa region of the Western blot is shown. D, interaction map of IsdC. The color code is the same as that described in the legend of Fig. 2. The dashed red line indicates functional segregation among the residues tested.

FIGURE 4.

Role of β7/β8 strand in heme transfer to IsdE. A, comparison between the structure of the β7/β8 strand of IsdC and that of IsdA. NEAT domains of IsdC and IsdA are depicted in green and red, respectively. Heme is shown in dark blue. The magenta sphere corresponds to the iron atom. The axial ligand Tyr-132 of IsdC is shown in cyan. B, amino acid sequences of the β7/β8 strand of IsdC, IsdA, and a mutant version of IsdA containing the underlined sequence of IsdC (so-called IsdA long loop). Asterisks indicate the position of the conserved tyrosine ligand. C, heme transfer from wild-type IsdA to IsdE. D, heme transfer from IsdA long loop to IsdE. E, kinetics of heme transfer to IsdE from IsdC (solid line), IsdA long loop (dashed line), and wild-type IsdA (dotted-dashed line) monitored at 411 nm. F, concentration dependence of the observed transfer rate of heme (k_obs) from IsdA long loop to IsdE.

FIGURE 5.

Heme binding and transfer in IsdE muteins. A, heme-binding properties of IsdE muteins. The spectra of wild-type IsdE and muteins were collected after incubation with heme and removal of excess ligand in a DEAE column. Green, magenta, and orange curves correspond to wild-type IsdE and muteins M78A and H229A, respectively. B, spectra of mixtures of holo-IsdC at 1.5 μm and apo-IsdE at 15 μm after a 30-min incubation. The color code is same as that described for A. The blue curve corresponds to holo-IsdC only. C, photo-cross-linking experiment with IsdC mutein A96X (containing pBPA) and IsdE mutein H229A. The arrow points to the photo-cross-linked product.

FIGURE 6.

Model of heme transport within cell wall of S. aureus. A, model without considering the self-transfer reaction. B, modified model considering self-transfer reactions in IsdA and IsdC. The arrows represent movement of heme among Isd transporters. The three types of cell-sorting motifs encountered in Isd proteins are shown as yellow (sortase A), blue (sortase B), and green (plasma membrane) anchors.

FIGURE 7.

Model of heme transfer between IsdC and IsdE. A, possible route of the heme moiety during its transfer from holo-IsdC to apo-IsdE. B, schematic view of the interaction. This model is based on kinetic, photo-cross-linking, and nano-LC-MS/MS data. IsdC is shown in gray, and IsdE is shown in violet. Portions of the β5/β6 and β7/β8 strands of IsdC are colored green. Heme is shown in dark blue, and the iron atom is shown in magenta. On the right panel, heme (Hm) is shown in red. The dashed lines represent possible interactions between Lys-124 of IsdC and residues 59–61 of IsdE, consistent with nano-LC-MS/MS analysis of photo-cross-linked mutein K124X. Residues 50–58 of IsdE, also detected in the MS/MS experiment, are not highlighted because they are buried within the IsdE structure or are far from the heme-binding pocket (>25 Å). The coordinates of IsdE were retrieved from the Protein Data Bank (code 2Q8Q) (24).