Structural investigation of IsdB, the hemoglobin receptor of Staphylococcus aureus
Fonner, Brittany Anne.
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Staphylococcus aureus is an opportunistic pathogen which when left unchallenged can cause severe toxicity and death in mammals. Critical to S. aureus growth is the ability to scavenge iron from hemoglobin (Hb). To acquire iron S. aureus has evolved a sophisticated protein-mediated heme acquisition pathway, which comprises nine iron-regulated surface determinant (Isd) proteins involved in heme capture, transport and degradation. A key protein of the acquisition pathway is the hemoglobin receptor protein IsdB, which comprises two NEAr transporter (NEAT) domains that act in concert to bind Hb and extract heme for subsequent transfer to downstream acquisition pathway proteins. Despite significant advances in the structural knowledge of other Isd proteins, the mechanisms and molecular basis of the IsdB-mediated heme acquisition process is not well understood. In order to provide more insights into the mode of function of IsdB, structural studies via nuclear magnetic resonance (NMR) spectroscopy were employed on different domains of IsdB. The three-dimensional solution structure of IsdBN1 revealed an immunoglobulin-like fold that is consistent with other NEAT domain proteins. Site directed mutagenesis studies revealed two key aromatic residues, F164 and Y167, involved in methemoglobin (metHb) interactions with IsdB. The protein variant F164D did not bind to metHb under NMR conditions. In heme transfer studies between metHb and IsdB constructs containing the two NEAT domains and the linker region, the amino acid substitution of F164D diminished but did not knock out the ability of IsdB to remove heme from metHb. A double amino acid substation of F164D and Y167D did abolish heme transfer from metHb to IsdB, therefore identifying key residues of IsdBN1 interaction with metHb. Studies of the linker region revealed an overall alpha-helical propensity and an interaction between the linker region and the second NEAT domain, IsdBN2. Solving the apo-IsdBN2 structure revealed slight differences in the heme-binding pocket, specifically in beta-strands 7 and 8 that interact with the heme moiety, when compared to the published crystal structure of holo-IsdBN2. The findings in this thesis provide a structural role for IsdBN1 enhancing the rate of extraction of heme from metHb by IsdBN2 and interactions between the domains of IsdB.