Structure-function analyses reveal key features in Staphylococcus aureus IsdB-associated unfolding of the heme-binding pocket of human hemoglobin.

IsdB is a receptor on the surface of the bacterial pathogen Staphylococcus aureus that extracts heme from hemoglobin (Hb) to enable growth on Hb as a sole iron source. IsdB is critically important both for in vitro growth on Hb and in infection models and is also highly up-regulated in blood, serum, and tissue infection models, indicating a key role of this receptor in bacterial virulence. However, structural information for IsdB is limited. We present here a crystal structure of a complex between human Hb and IsdB. In this complex, the α subunits of Hb are refolded with the heme displaced to the interface with IsdB. We also observe that atypical residues of Hb, His58 and His89 of αHb, coordinate to the heme iron, which is poised for transfer into the heme-binding pocket of IsdB. Moreover, the porphyrin ring interacts with IsdB residues Tyr440 and Tyr444 Previously, Tyr440 was observed to coordinate heme iron in an IsdB·heme complex structure. A Y440F/Y444F IsdB variant we produced was defective in heme transfer yet formed a stable complex with Hb (Kd = 6 ± 2 µm) in solution with spectroscopic features of the bis-His species observed in the crystal structure. Haptoglobin binds to a distinct site on Hb to inhibit heme transfer to IsdB and growth of S. aureus, and a ternary complex of IsdB·Hb·Hp was observed. We propose a model for IsdB heme transfer from Hb that involves unfolding of Hb and heme iron ligand exchange.

Hemoglobin binding and catalytic heme extraction by IsdB near iron transporter domains.

The Isd (iron-regulated surface determinant) system is a multiprotein transporter that allows bacterium Staphylococcus aureus to take up iron from hemoglobin (Hb) during human infection. In this system, IsdB is a cell wall-anchored surface protein that contains two near iron transporter (NEAT) domains, one of which binds heme. IsdB rapidly extracts heme from Hb and transfers it to IsdA for relay into the bacterial cell. Using a series of recombinant IsdB constructs that included at least one NEAT domain, we demonstrated that both domains are required to bind Hb with high affinity (KD = 0.42 ± 0.05 µM) and to extract heme from Hb. Moreover, IsdB extracted heme only from oxidized metHb, although it also bound oxyHb and the Hb-CO complex. In a reconstituted model of the biological heme relay pathway, IsdB catalyzed the transfer of heme from metHb to IsdA with a Km for metHb of 0.75 ± 0.07 µN and a kcat of 0.22 ± 0.01 s(-1). The latter is consistent with the transfer of heme from metHb to IsdB being the rate-limiting step. With both NEAT domains and the linker region present in a single contiguous polypeptide, high-affinity Hb binding was achieved, rapid heme uptake was observed, and multiple turnovers of heme extraction from metHb and transfer to IsdA were conducted, representing all known Hb-heme uptake functions of the full-length IsdB protein.