The IsdB Unfolding of Heme-Binding Pocket of Human Hemoglobin

The IsdB Unfolding of Heme-Binding Pocket of Human Hemoglobin

            Staphylococcus aureus (S. aureus) is a member of human flora, colonizing the nostrils and hands. The bacterial pathogen’s survival in the host is dependent on the availability of iron (Mikkelsen et al. 1782). When the pathogen senses low iron, it initiates a surface iron-acquisition program (IsdB), enabling the capture of haptoglobin and heme from hemoglobin (Bowden et al. 177). The IsdB iron receptor plays an essential role in the pathogen’s virulence. S. aureus is the most common cause of nosocomial infections. It also causes many human diseases, such as boils and severe blood infections (Mikkelsen et al. 1785). The bacteria require an effective system to scavenge iron from the host because the human body highly monitors its trafficking (Bowden et al. 177). The monitoring of the body’s iron levels is to limit microbial growth and minimize the toxicity resulting from free iron (Bowden et al. 177). The “iron-regulated surface determinant” (Isd) system is made up of nine components with four surface proteins. The surface proteins are covalently bonded to peptidoglycan that binds the heme, that is, IsdABCH. IsdH and IsdB are the only components of the Isd system that can attach to hemoproteins (Bowden et al. 178). IsdH is capable of binding hemoglobin (Hb), haptoglobin (Hp), and the Hb.Hp complex. Hp on its own does not constitute an iron source but instead hinders the extraction of heme from the IsdB^N1N2.Hb complex.

Heme was positioned between IsdH and IsdB that were consistent with the intermediate state of the transfer of heme from hemoglobin. This followed the introduction of two-point mutations into the IsdB^N1N2 so that it could trap any similar species available in the solution. The solution was treated with 2.1-2.4 Molar ammonium sulfate and citric acid to maintain a pH of between 5.0-5.5. The solution was left to stand for four weeks. Characterization was done spectroscopically to determine the IsdB^N1N2.Hb complex crystal structure. Kinetic analyses were also conducted to investigate the heme transfer process.

The IsdB^N1N2.Hb complex showed four IsdB molecules asymmetrically surrounding a central hemoglobin (Bowden et al. 181). Each IsdB^N1N2 molecule structure resembles a dumb-bell, which has a pair of NEAT domains that are linked by an alpha-helical linker, as shown in figure 2A. This was also observed for the IsdB^N2N3 molecule. The missing IsdB components in the modeling would cause a tremendous steric clash, suggesting that proteolysis allowed the crystallization process. The hemoglobin chains indicate significant structural rearrangements as opposed to the complex developed in isolated oxy-hemoglobin. In this crystal structure, heme is sandwiched in a pocket between helices F and E, where the F helix coordinated directly to the E helix and the heme iron (Bowden et al. 181). After binding to the IsdB^N1N2, there is a significant deformation of the alpha-hemoglobin heme pocket. A portion of the E helix and the F helix of each alpha-hemoglobin chain unwinds entirely (Bowen et al. 179). A double-mutant IsdB^N1N2 complex that replaced Tyr440 with Phe prevented the transfer of heme. There was a significant transfer of heme from met-hemoglobin to IsdB^N1N2. This trend was investigated using stopped-flow spectroscopy marked by a considerable intensity shift between 350nm and 420nm (Bowden et al. 183). It was also evident that Hp hindered the transfer of heme from met-hemoglobin to IsdB^N1N2. The Hp binds with the met-hemoglobin released to stop oxidative damage.

The transfer of heme by Staphylococcus aureus surface receptors from body cells was investigated under controlled conditions. IsdB and IsdH capabilities to retrieve heme that is needed by the bacteria was investigated. The resulting IsdB^N1N2.Hb complex crystals were investigated using light spectroscopy, and it was found that four molecules of IsdB surrounded a central hemoglobin molecule in an asymmetrical configuration. Hemoglobin chains indicated significant structural rearrangements compared with the crystal structures formed with oxy-hemoglobin. This means that Staphylococcus aureus IsdB cannot transfer heme from oxyHb. It was also found that Hp hinders the movement of heme by replacing Try⁴⁴⁰ with Phe that inhibits the movement of heme from met-hemoglobin to IsdB^N1N2.

Works Cited

Bowden, Catherine FM, et al. “Structure-function analyses reveal key features in Staphylococcus aureus IsdB-associated unfolding of the heme-binding pocket of human hemoglobin.” Journal of Biological Chemistry 293.1 (2018): 177-190.

Mikkelsen, Jakob H., Kasper Runager, and Christian BF Andersen. “The human protein haptoglobin inhibits IsdH-mediated heme-sequestering by Staphylococcus aureus.” Journal of Biological Chemistry 295.7 (2020): 1781-1791.