The Staphylococcus aureus IsdH Receptor Forms a Dynamic Complex with Human Hemoglobin that Triggers Heme Release via Two Distinct Hot Spots

• Published in: Journal of molecular biology 2020-02, Vol.432 (4), p.1064-1082
• Main Authors: Ellis-Guardiola, Ken, Clayton, Joseph, Pham, Clarissa, Mahoney, Brendan J., Wereszczynski, Jeff, Clubb, Robert T.
• Format: Article
• Language: English
• Subjects: Antigens, Bacterial - chemistry; Antigens, Bacterial - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Heme - metabolism; Hemoglobins - chemistry; Hemoglobins - metabolism; Humans; Iron - metabolism; Iron-regulated surface determinant system; Magnetic Resonance Spectroscopy; Molecular dynamics; Molecular Dynamics Simulation; Nuclear magnetic resonance spectroscopy; Protein Binding; Protein Structure, Secondary; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - metabolism; Spectrophotometry; Staphylococcus aureus - metabolism; Staphylococcus aureus hemoglobin receptor; Stopped-flow spectrophotometry
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2019.12.023

Iron is an essential nutrient that is actively acquired by bacterial pathogens during infections. Clinically important Staphylococcus aureus obtains iron by extracting heme from hemoglobin (Hb) using the closely related IsdB and IsdH surface receptors. In IsdH, extraction is mediated by a conserved tridomain unit that contains its second (N2) and third (N3) NEAT domains joined by a helical linker, called IsdHN2N3. Leveraging the crystal structure of the IsdHN2N3:Hb complex, we have probed the mechanism of heme capture using NMR, stopped-flow transfer kinetics measurements, and molecular dynamics (MD) simulations. NMR studies of the 220 kDa IsdHN2N3:Hb complex reveal that it is dynamic, with persistent interdomain motions enabling the linker and N3 domains in the receptor to transiently engage Hb to remove its heme. An alanine mutagenesis analysis reveals that two receptor subsites positioned ~20 Å apart trigger heme release by contacting Hb's F-helix. These subsites are located within the N3 and linker domains and appear to play distinct roles in stabilizing the heme transfer transition state. Linker domain contacts primarily function to destabilize Hb–heme interactions, thereby lowering ΔH‡, while contacts from the N3 subsite play a similar destabilizing role, but also form a bridge through which heme moves from Hb to the receptor. Interestingly, MD simulations suggest that within the transiently forming interface, both the F-helix and receptor bridge are in motion, dynamically sampling conformations that are suitable for heme transfer. Thus, IsdH triggers heme release from Hb via a flexible, low-affinity interface that forms fleetingly in solution. [Display omitted] •220 kDa complex between Hb and IsdH retains interdomain flexibility in solution.•Alanine scanning highlights two hot spots responsible for heme extraction.•IsdH transiently stabilizes distorted conformation of Hb F-helix.•MD reveals dynamic formation of a heme-transfer bridge between Hb and IsdH.•IsdH triggers heme release through flexible, low-affinity interface with Hb.